Methods of diagnosing risk of myocardial infarction by detection of polymorphisms in connexin and NADH/NADPH oxidase genes

ABSTRACT

It is intended to provide a means of diagnosing myocardial infarction which shows a high accuracy and a high predictability. The risk of myocardial infarction is diagnosed by a method comprising the following steps: (i) the step of analyzing 2 or more polymorphisms among 10 gene polymorphisms or 5 gene polymorphisms proved as relating to myocardial infarction; (ii) the step of determining the genotype of a nucleic acid sample based on the polymorphism data obtained in the above step; and (iii) the step of determining the genetic risk of myocardial infarction from the genotype thus obtained.

TECHNICAL FIELD

The present invention relates to a detection method using genesassociated with myocardial infarction. More particularly, it relates toa detection method using a plurality of gene polymorphisms associatedwith myocardial infarction and to a kit used for the method. The presentinvention can be used for, for example, diagnosing a risk of myocardialinfarction.

BACKGROUND ART

Myocardial infarction is a multifactorial disease and its development isdefined by the interaction between individual's genetic background andvarious environmental factors (Marenberg M E, Risch N, Berkman L F,Floderus B, de Faire U. Genetic susceptibility to death from coronaryheart disease in a study of twins. N Engl J Med 1994;330:1041-1046.,Nora J J, Lortscher R H, Spangler R D, Nora A H, Kimberling W J.Genetic-epidemiologic study of early-onset ischemic heart disease.Circulation 1980; 61: 503-508). In general, the risk of myocardialinfarction is increased in proportion to the number of conventional riskfactors such as hypertension, diabetes, hyperlipidemia, etc (Nora J J,Lortscher R H, Spangler R D, Nora A H, Kimberling W J.Genetic-epidemiologic study of early-onset ischemic heart disease.Circulation 1980; 61: 503-508). These risk factors themselves arepartially regulated by genetic factors. However, since a family historyis an independent predictable factor of myocardial infarction, it issuggested that there are genetic factors susceptible for myocardialinfarction other than conventional risk factors (Marenberg M E, Risch N,Berkman L F, Floderus B, de Faire U. Genetic susceptibility to deathfrom coronary heart disease in a study of twins. N Engl J Med 1994; 330:1041-1046). In addition, cases in which myocardial infarction may bedeveloped without any conventional risk factors also suggest therelationship between myocardial infarction and genetic factors.

Myocardial infarction is a disease with the highest mortality in theWestern countries. Even in the case where myocardial infarction is notlethal, it may be complicated with heart failure, angina pectoris andrefractory arrhythmia, thus deteriorating the quality of life ofpatients. Therefore, needless to say, it is important to preventmyocardial infarction. One of the methods for preventing myocardialinfarction is to identify genes susceptible for myocardial infarction.By linkage analysis (Broeckel U, Hengstenberg C, Mayer B, et al. Acomprehensive linkage analysis for myocardial infarction and its relatedrisk factors. Nature genet 2002;30:210-214) and association studies bycandidate genes (Cambien F, Poirier O, Lecerf L, et al. Deletionpolymorphism in the gene for angiotensin-converting enzyme is a potentrisk factor for myocardial infarction. Nature 1992; 359: 641-644. WeissE J, Bray P F, Tayback M, et al. A polymorphism of a plateletglycoprotein receptor as an inherited risk factor for coronarythrombosis. N Engl J Med 1996; 334: 1090-1094., Iacoviello L, DiCastelnuovo A, De Knijff P, et al. Polymorphisms in the coagulationfactor VII gene and the risk of myocardial infarction. N Engl J Med1998;338:79-85., Kuivenhoven J A, Jukema J W, Zwinderman A H, et al. Therole of a common variant of the cholesterol ester transfer protein genein the progression of coronary atherosclerosis. N Engl J Med 1998; 338:86-93), gene locus on the chromosome and some candidate genes which areassociated with myocardial infarction have been identified. Previously,by studies of genetic epidemiology, there have been reported therelationships between the myocardial infarction and gene polymorphismssuch as angiotensin converting enzyme (Cambien F, Poirier O, Lecerf L,et al. Deletion polymorphism in the gene for angiotensin-convertingenzyme is a potent risk factor for myocardial infarction. Nature 1992;359: 641-644), platlet glycoprotein IIIa (Weiss E J, Bray P F, TaybackM, et al. A genetical polymorphism of a platelet glycoprotein receptoras an inherited risk factor for coronary thrombosis. N Engl J Med 1996;334: 1090-1094), blood coagulation factor VII, cholesterol estertnrasfer protein (Kuivenhoven J A, Jukema J W, Zwinderman A H, et al.The role of a common variant of the cholesterol ester transfer proteingene in the progression of coronary atherosclerosis. N Engl J Med 1998;338: 86-93), and the like. However, there have been conflicting reports,certain conclusion has never been reached. Furthermore, since differentraces have different gene polymorphisms, it is important to construct adatabase as to the relationship between polymorphisms and myocardialinfarction in each race.

DISCLOSURE OF INVENTION

As mentioned above, a large number of relation analyses between genepolymorphisms and coronary artery disease or myocardial infarction havebeen carried out previously. However, many studies have not reached acertain finding in terms of significance thereof. This is mainly becausepopulations of subjects in many studies are not sufficient and not onlygene polymorphisms but also environmental factors are different betweenraces. Furthermore, even if the relationship with myocardial infarctionis recognized, in the analysis of large scale population, relative risk(odds ratio) is generally low.

The present invention was made on the basis of the above-mentionedbackground, and the object thereof is to provide a means of diagnosinggenetic risk of myocardial infarction with high accuracy and highpredictability and to contribute to a primary prevention of myocardialinfarction.

To achieve the above-mentioned objects, the present inventors haveextracted 71 genes which were estimated to be associated with coronaryarteriosclerosis, coronary artery spasm, hypertension, diabetes,hyperlipidemia, etc., and mainly selected 112 polymorphisms which werepredicted to be associated with functional changes of genes by the useof a plurality of public databases. Then, as to 112 polymorphisms of 71genes, a large scale relation analysis of more than 5000 cases wascarried out. As a result, the present inventors succeeded in identifyingten SNPs (single nucleotide polymorphisms) which were associated withmyocardial infarction in males and five SNPs in females. In addition, byusing the combination of these polymorphisms, it was found that in astepwise forward selection of multivariate logistic regression analysis,a maximum odds ratio of 11.26 in males and maximum odds ratio of 88.51in females were presented. Based on these results, it was possible toobtain a finding in that by selecting a plurality of SNPs from theseSNPs and using the combination of the results of analysis of each SNP,diagnosis of myocardial infarction can be carried out with highreliability and high predictability. Meanwhile as to one of the fiveSNPs that were found to be associated with myocardial infarction infemales, even by analyzing the polymorphism singly, extremely high oddsratio could be obtained. The present invention was completed based onthe above findings and provides the following configuration.

-   [1] A method for detecting the genotype in a nucleic acid sample,    comprising the following step (a):    -   (a) analyzing two or more polymorphisms selected from the group        consisting of the following (1) to (10) in a nucleic acid        sample:        -   (1) polymorphism at the base number position 1019 of the            connexin 37 gene;        -   (2) polymorphism at the base number position −863 of the            tumor necrosis factor α gene;        -   (3) polymorphism at the base number position 242 of the            NADH/NADPH oxidase p22 phox gene;        -   (4) polymorphism at the base number position −6 of the            angiotensinogen gene:        -   (5) polymorphism at the base number position −219 of the            apolipoprotein E gene;        -   (6) polymorphism at the base number position 994 of the            platelet-activating factor acetylhydrolase gene;        -   (7) polymorphism at the base number position −482 of the            apolipoprotein C-III gene;        -   (8) polymorphism at the base number position 1186 of the            thrombospondin 4 gene;        -   (9) polymorphism at the base number position −819 of the            interleukin-10 gene; and        -   (10) polymorphism at the base number position −592 of the            interleukin-10 gene.-   [2] A method for detecting the genotype in a nucleic acid sample,    comprising the following step (b):    -   (b) analyzing two or more polymorphisms selected from the group        consisting of the following (11) to (15) in a nucleic acid        sample:        -   (11) polymorphism at the base number position −1171 of the            stromelysin 1 gene;        -   (12) polymorphism at the base number position −668 of the            plasminogen activator inhibitor-1 gene;        -   (13) polymorphism at the base number position 1018 of the            glycoprotein Ibα gene;        -   (14) polymorphism at the base number position 584 of the            paraoxonase gene: and        -   (15) polymorphism at the base number position 4070 of the            apolipoprotein E gene.-   [3] A method for detecting the genotype in a nucleic acid sample,    comprising the following step (c):    -   (c) analyzing polymorphism at the base number position 4070 of        the apolipoprotein E gene in a nucleic acid sample.-   [4] A method for diagnosing the risk of myocardial infarction,    comprising the following steps (i) to (iii):    -   (i) analyzing two or more polymorphisms selected from the group        consisting of the following (1) to (10) in a nucleic acid        sample:        -   (1) polymorphism at the base number position 1019 of the            connexin 37 gene;        -   (2) polymorphism at the base number position −863 of the            tumor necrosis factor α gene;        -   (3) polymorphism at the base number position 242 of the            NADH/NADPH oxidase p22 phox gene;        -   (4) polymorphism at the base number position −6 of the            angiotensinogen gene:        -   (5) polymorphism at the base number position −219 of the            apolipoprotein E gene;        -   (6) polymorphism at the base number position 994 of the            platelet-activating factor acetylhydrolase gene;        -   (7) polymorphism at the base number position −482 of the            apolipoprotein C-III gene;        -   (8) polymorphism at the base number position 1186 of the            thrombospondin 4 gene;        -   (9) polymorphism at the base number position −819 of the            interleukin-10 gene; and        -   (10) polymorphism at the base number position −592 of the            interleukin-10 gene;    -   (ii) determining, based on the information about polymorphism        which was obtained in the step (i), the genotype of the nucleic        acid sample; and    -   (iii) assessing, based on the genotype determined, a genetic        risk of myocardial infarction.-   [5] A method for diagnosing the risk of myocardial infarction,    comprising the following steps (iv) to (vi):    -   (iv) analyzing two or more polymorphisms selected from the group        consisting of the following (11) to (15) in a nucleic acid        sample:        -   (11) polymorphism at the base number position −1171 of the            stromelysin 1 gene;        -   (12) polymorphism at the base number position −668 of the            plasminogen activator inhibitor-1 gene;        -   (13) polymorphism at the base number position 1018 of the            glycoprotein Ibα gene;        -   (14) polymorphism at the base number position 584 of the            paraoxonase gene: and        -   (15) polymorphism at the base number position 4070 of the            apolipoprotein E gene;    -   (v) determining, based on the information about polymorphism        which was obtained in the step (i), the genotype of the nucleic        acid sample; and    -   (vi) assessing, based on the genotype determined, a genetic risk        of myocardial infarction.-   [6] A method for diagnosing the risk of myocardial infarction,    comprising the following steps (vii) to (ix):    -   (vii) analyzing polymorphism at the base number position 4070 of        the apolipoprotein E gene in a nucleic acid sample;    -   (viii) determining, based on the information about polymorphism        which was obtained in the step (vii), the genotype of the        nucleic acid sample; and    -   (ix) assessing, based on the genotype determined, a genetic risk        of myocardial infarction.-   [7] A kit for detecting the genotype, comprising two or more of    nucleic acids selected from the group consisting of the    following (1) to (10):    -   (1) a nucleic acid for polymorphism analysis at the base number        position 1019 of the connexin 37 gene;    -   (2) a nucleic acid for polymorphism analysis at the base number        position −863 of the tumor necrosis factor α gene;    -   (3) a nucleic acid for polymorphism analysis at the base number        position 242 of the NADH/NADPH oxidase p22 phox gene;    -   (4) a nucleic acid for polymorphism analysis at the base number        position −6 of the angiotensinogen gene:    -   (5) a nucleic acid for polymorphism analysis at the base number        position −219 of the apolipoprotein E gene;    -   (6) a nucleic acid for polymorphism analysis at the base number        position 994 of the platelet-activating factor acetylhydrolase        gene;    -   (7) a nucleic acid for polymorphism analysis at the base number        position −482 of the apolipoprotein C-III gene;    -   (8) a nucleic acid for polymorphism analysis at the base number        position 1186 of the thrombospondin 4 gene;    -   (9) a nucleic acid for polymorphism analysis at the base number        position −819 of the interleukin-10 gene; and    -   (10) a nucleic acid for polymorphism analysis at the base number        position −592 of the interleukin-10 gene.-   [8] A kit for detecting the genotype, comprising two or more of    nucleic acids selected from the group consisting of the    following (11) to (15):    -   (11) a nucleic acid for polymorphism analysis at the base number        position −1171 of the stromelysin 1 gene;    -   (12) a nucleic acid for polymorphism analysis at the base number        position −668 of the plasminogen activator inhibitor-1 gene;    -   (13) a nucleic acid for polymorphism analysis at the base number        position 1018 of the glycoprotein Ibα gene;    -   (14) a nucleic acid for polymorphism analysis at the base number        position 584 of the paraoxonase gene: and    -   (15) a nucleic acid for polymorphism analysis at the base number        position 4070 of the apolipoprotein E gene.-   [9] A kit for detecting the genotype, comprising a nucleic acid for    polymorphism analysis at the base number position 4070 of the    apolipoprotein E gene.-   [10] Fixed nucleic acids comprising the following two or more    nucleic acid selected from the group consisting of the following (1)    to (10) fixed to an insoluble support:    -   (1) a nucleic acid for polymorphism analysis at the base number        position 1019 of the connexin 37 gene;    -   (2) a nucleic acid for polymorphism analysis at the base number        position −863 of the tumor necrosis factor α gene;    -   (3) a nucleic acid for polymorphism analysis at the base number        position 242 of the NADH/NADPH oxidase p22 phox gene;    -   (4) a nucleic acid for polymorphism analysis at the base number        position −6 of the angiotensinogen gene:    -   (5) a nucleic acid for polymorphism analysis at the base number        position −219 of the apolipoprotein E gene;    -   (6) a nucleic acid for polymorphism analysis at the base number        position 994 of the platelet-activating factor acetylhydrolase        gene;    -   (7) a nucleic acid for polymorphism analysis at the base number        position −482 of the apolipoprotein C-III gene;    -   (8) a nucleic acid for polymorphism analysis at the base number        position 1186 of the thrombospondin 4 gene;    -   (9) a nucleic acid for polymorphism analysis at the base number        position −819 of the interleukin-10 gene; and    -   (10) a nucleic acid for polymorphism analysis at the base number        position −592 of the interleukin-10 gene.-   [11] Fixed nucleic acids comprising the following two or more    nucleic acid selected from the group consisting of the    following (11) to (15) fixed to an insoluble support:    -   (11) a nucleic acid for polymorphism analysis at the base number        position −1171 of the stromelysin 1 gene;    -   (12) a nucleic acid for polymorphism analysis at the base number        position −668 of the plasminogen activator inhibitor-1 gene;    -   (13) a nucleic acid for polymorphism analysis at the base number        position 1018 of the glycoprotein Ibα gene;    -   (14) a nucleic acid for polymorphism analysis at the base number        position 584 of the paraoxonase gene: and    -   (15) a nucleic acid for polymorphism analysis at the base number        position 4070 of the apolipoprotein E gene.-   [12] Fixed nucleic acids comprising a nucleic acid for polymorphism    analysis at the base number position 4070 of the apolipoprotein E    gene fixed to an insoluble support.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a table summarizing 112 gene polymorphisms examined in ascreening related analysis in Examples.

FIG. 2 is also a table summarizing 112 gene polymorphisms examined in ascreening related analysis in Examples.

FIG. 3 is a table summarizing primers (SEQ ID NOs: 30, 31, 32, 21, 22,23, 15, 16, 17, 24, 25, 26, 18, 19, 20, 33, 34, 35, 42, 43 and 44 inthis order from the top), probes (SEQ ID NOs: 59 and 60 in this orderfrom the top) and other conditions used in order to determine thegenotype in Examples. In FIG. 3, FITC denotes fluorescein isothiocyanateand TxR denotes Texas Red, respectively.

FIG. 4 is also a table summarizing primers (SEQ ID NOs: 27, 28, 29, 39,40, 41, 36, 37, 38, 53, 54, 55, 56, 57, 58, 48, 49, 45, 46, 47, 50, 51and 52 in this order from the top), probes (SEQ ID NOs: 61, 62, 63 and64 in this order from the top) and other conditions used in order todetermine the genotype in Examples. In FIG. 4, FITC denotes fluoresceinisothiocyanate and TxR denotes Texas Red, respectively.

FIG. 5 is a table summarizing the backgrounds of 909 gene polymorphismsexamined in a screening related analysis in Examples. Data of age andbody mass index are represented by average i standard deviation. Intable, *1 denotes P=0.0278 and *2 denotes P<0.0001 versus controls,respectively.

FIG. 6 is a table summarizing gene polymorphisms which were found to beassociated with myocardial infarction.

FIG. 7 is a table summarizing the backgrounds of all 5061 subjects inthe relation analysis in Examples. Data of age and body mass index arerepresented by average ± standard deviation. In table, *1 denotesP=0.022, *2 denotes P<0.001 and *3 denotes P=0.017, respectively.

FIG. 8 is a table summarizing distribution of gene polymorphisms whichwere found to be associated with myocardial infarction in all 5061subjects in the relation analysis in Examples.

FIG. 9 is a table showing results of multivariate logistic regressionanalysis of gene polymorphisms and myocardial infarction in all 5061subjects in the relation analysis according to Examples. In table, ORdenotes odds ratio and CI denotes confidence interval.

FIG. 10 is a table showing results of stepwise forward selection methodof multivariate logistic regression analysis of gene polymorphismsassociated with myocardial infarction. In this table, CI denotesconfidence interval.

FIG. 11 is a table showing results of diagnosis of genetic risk (risk ofdevelopment) of myocardial infarction using a combination of 5 genepolymorphisms in male.

FIG. 12 is a table showing results of diagnosis of genetic risk (risk ofdevelopment) of myocardial infarction using a combination of 5 genepolymorphisms in female.

FIG. 13 is a graph showing the relationship between the number of genepolymorphisms combined and the odds ratio in contracting myocardialinfarction. Note here that (A) shows the case of male subjects and (B)shows the case of female subjects.

BEST MODE FOR CARRYING OUT THE INVENTION

The first aspect of the present invention relates to a method fordetecting the genotype in a nucleic acid sample. One embodiment of thepresent invention is featured by including the step of analyzing two ormore polymorphisms selected from the group consisting of the following(1) to (10). Furthermore, another embodiment of the present invention isfeatured by including the step of analyzing two or more polymorphismsselected from the group consisting of the following (11) to (15). Inaddition, further embodiment of the present invention is featured byincluding at least the step of analyzing the following (15). Note herethat it is possible to determine, based on the information aboutpolymorphism which was obtained in the above-mentioned step, thegenotype of the nucleic acid sample; and thereby to assess, based on thegenotype determined, a genetic risk of myocardial infarction.

-   -   (1) polymorphism at the base number position 1019 of the        connexin 37 gene: 1019C→T (hereinafter, also referred to as        “connexin 37 (1019C→T) polymorphism”)    -   (2) polymorphism at the base number position −863 of the tumor        necrosis factor α gene: −863C→A (hereinafter, also referred to        as “TNFα (−863C→A) polymorphism”)    -   (3) polymorphism at the base number position 242 of the        NADH/NADPH oxidase p22 phox gene: 242C→T (hereinafter, also        referred to as “NADH/NADPH oxidase p22 phox (242C→T)        polymorphism”)    -   (4) polymorphism at the base number position −6 of the        angiotensinogen gene: −6→GA (hereinafter, also referred to as        “polymorphism angiotensinogen (−6G→A)”)    -   (5) polymorphism at the base number position −219 of the        apolipoprotein E gene: −219G→T (hereinafter, also referred to as        “Apo E-219 (−219G→T) polymorphism”)    -   (6) polymorphism at the base number position 994 of the        platelet-activating factor acetylhydrolase gene: 994G→T        (hereinafter, also referred to as “PAF acetylhydrolase (994G→T)        polymorphism”)    -   (7) polymorphism at the base number position −482 of the        apolipoprotein C-III gene: −482C→T (hereinafter, also referred        to as “Apo C-III (−482C→T) polymorphism”)    -   (8) polymorphism at the base number position 1186 of the        thrombospondin 4 gene: 1186G→(hereinafter, also referred to as        “TSP4 (1186G→C) polymorphism”)    -   (9) polymorphism at the base number position −819 of the        interleukin-10 gene: −819T→C (hereinafter, also referred to as        “IL-10 (−819T→C) polymorphism”)    -   (10) polymorphism at the base number position −592 of the        Interleukin-10 gene: −592A→C (hereinafter, also referred to as        “IL-10 (−592A→C) polymorphism”)    -   (11) polymorphism at the base number position −1171 of the        stromelysin 1 gene: −1171/5A→6A (hereinafter, also referred to        as “stromelysin 1 (−1171/5A→6A) polymorphism”)    -   (12) polymorphism at the base number position −668 of the        plasminogen activator inhibitor 1 gene: −668/4G→5G (hereinafter,        also referred to as “PAI1 (−668/4G→5G) polymorphism”)    -   (13) polymorphism at the base number position 1018 of the        glycoprotein Ibα gene: 1018C→T (hereinafter, also referred to as        “glycoprotein Ibα (1018C→T) polymorphism”)    -   (14) polymorphism at the base number position 584 of the        paraoxonase gene: 584G→A (hereinafter, also referred to as        “paraoxonase (584G→A) polymorphism”)    -   (15) polymorphism at the base number position 4070 of the        apolipoprotein E gene: 4070C→T (hereinafter, also referred to as        “Apo E (4070C→T) polymorphism”)

In the above, description such as 1019C→T means that polymorphism at therelevant base number position consists of two genotypes, bases beforeand after the arrow. Herein, −1171/5A→6A means a polymorphism consistingof a genotype having five A (adenines) existing successively in the 3′direction from the base number position −1171 and a genotype having sixA existing successively in the 3′ direction from the base numberposition −1171. Similarly, −668/4G→5G means a polymorphism consisting ofa genotype having four G (guanines) existing successively in the 3′direction from the base number −668 and a gene having five G existingsuccessively in the 3′ direction from the base number −668.

The base number of each gene is expressed using as standards the knownsequences which are registered in the public database, GenBank (NCBI).Note here that in the base sequence of SEQ ID NO: 1 (Accession No.M96789: Homo sapiens connexin 37 (GJA4) mRNA, complete cds), the 1019thbase corresponds to the base at position 1019 of the connexin 37 gene.Similarly, in the base sequence of SEQ ID NO: 2 (Accession No. L11698:Homo sapiens tumor necrosis factor alpha gene, promoter region), the197th base corresponds to the base at position −863 of tumor necrosisfactor α gene; in the base sequence of SEQ ID NO: 3 (Accession No.M61107: Homo sapiens cytochrome b light chain (CYBA) gene, exons 3 and4), the 684th base corresponds to the base at position 242 of NADH/NADPHoxidase p22 phox gene; in the base sequence of SEQ ID NO: 4 (AccessionNo. X15323: H. apiens angiotensinogen gene 5′ region and exon 1), the463th base corresponds to the base at position −6 of angiotensinogengene; in the base sequence of SEQ ID NO: 5 (Accession No. AF055343: Homosapiens apolipoprotein E (APOE) gene, 5′ regulatory region, partialsequence), the 801th base corresponds to the base at position −219 ofthe apolipoprotein E gene; in the sequence of SEQ ID NO: 6 (AccessionNo. U20157: Human platelet-activating factor acetylhydrolase mRNA,complete cds), the 996th base corresponds to the base at position 994 ofthe platelet-activating factor acetylhydrolase gene; in the sequence ofSEQ ID NO: 7 (Accession No. X13367: Human DNA for apolipoprotein C-III5′-flank), the 936th base corresponds to the base at position −482 ofthe apolipoprotein C-III gene; in the sequence of SEQ ID NO: 8(Accession No. Z19585: H. sapiens mRNA for thrombospondin-4), the 1186thbase corresponds to the base at position 1186 of the thrombospondin 4gene; in the sequence of SEQ ID NO: 9 (Accession No. Z30175: H. sapiensIL-10 gene for interleukin-10 (promoter)), the 455th base corresponds tothe base at position −819 and the 682th base corresponds to the base atposition −592 of the interleukin-10 gene, respectively; in the sequenceof SEQ ID NO: 10 (Accession No. U43511: Homo sapiens stromelysin-1 gene,promoter region), the 698th base corresponds to the base at position−1171 of the stromelysin 1 gene; in the sequence of SEQ ID NO: 11(Accession No. X13323: Human gene for plasminogen activator inhibitor 1(PAI-1) 5′-flank and exon 1), the 131th base corresponds to the base atposition −668 of the plasminogen activator inhibitor 1 gene; in thesequence of SEQ ID NO: 12 (Accession No. J02940: Human plateletglycoprotein Ib alpha chain mRNA, complete cds), the 524th basecorresponds to the base at position 1018 of the glycoprotein Ibα gene;in the sequence of SEQ ID NO: 13 (Accession No. M63012: H. sapiens serumparaoxonase (PON) 1 mRNA, complete cds), the 584th base corresponds tothe base at position 584 of the paraoxonase gene; and in the sequence ofSEQ ID NO: 14 (Accession No. M10065: Human apolipoprotein E (epsilon-4allele) gene, complete cds), the 4070th base corresponds to the base atposition 4070 of the apolipoprotein E gene.

In the present invention, “analyzing polymorphism” refers to theinvestigation as to what genotype a nucleic acid sample has in the genepolymorphism to be analyzed. It is the same meaning as the investigationon the base (base sequence) of the position in which the polymorphismexists. Typically, for example, in the case of the analysis of theconnexin 37 (1019C→T) polymorphism, it refers to investigation on whatgenotype, i.e., TT (homozygote of allele T) or CT (heterozygote ofallele C and allele T) or CC (homozygote of allele C), the connexin 37gene in a nucleic acid sample has.

As shown in Examples mentioned below, the polymorphisms mentioned (1) to(10) above are polymorphisms that are recognized as being particularlyeffective to be used in determining genetic risk of myocardialinfarction in an analysis of Japanese male subjects. Therefore, analysistargeting these polymorphisms enables diagnosis with higher accuracy andwith higher predictability when subjects are males, particularly,Japanese males.

Similarly, as shown in Examples mentioned below, the polymorphismsmentioned (11) to (15) above are polymorphisms that are recognized asbeing particularly effective to be used in determining genetic risk ofmyocardial infarction in an analysis of Japanese female subjects.Therefore, analysis targeting these polymorphisms enables diagnosis withhigher accuracy and with higher predictability when subjects arefemales, particularly, Japanese females. Among these polymorphisms, asto the polymorphism described in (15), as shown in Examples mentionedbelow, it was confirmed that by analyzing the polymorphism, it waspossible to determine the genetic risk of myocardial infarction withextremely high odds ratio. Therefore, by analyzing this polymorphismsingly, it is possible to determine the genetic risk of myocardialinfarction with high accuracy and high predictability. Of course, inaddition to the analysis of the polymorphism (15), analysis of any oneor a plurality of polymorphisms selected from (11) to (14) is carriedout in combination, and thereby it may be possible to detect thegenotype or to diagnose a genetic risk of myocardial infarction.

Herein, in principle, in proportion to the increase in the number ofpolymorphisms to be analyzed, the genotypes of nucleic acid sample areclassified more finely. Thereby, it is possible to diagnose a geneticrisk of myocardial infarction with higher predictability. From thisviewpoint, it is preferable to detect the genotype by analyzing a largernumber of polymorphisms in the above-mentioned polymorphisms (1) to(10). Therefore, it is the most preferable to analyze all of thepolymorphisms (1) to (10). In the case where detection is carried out bycombining nine or less of polymorphisms, it is possible topreferentially select the polymorphisms with higher odds ratios as inExamples mentioned below. For example, in the case where eightpolymorphisms are used in combination, it is preferable to select ninepolymorphisms with higher odds ratio, that is, to select (1), (3), (5),(6), (7), (8), (9) and (10). Similarly, in the case where sevenpolymorphisms are used in combination, it is preferable to select (1),(3), (5), (6), (8), (9) and (10). Similarly, in the case where 6polymorphisms are used in combination, it is preferable to select (1),(5), (6), (8), (9) and (10). Similarly, in the case where fivepolymorphisms are used in combination, it is preferable to select (1),(5), (6), (8), and (9).

Similarly, in the case where two or more polymorphisms selected from thegroup consisting of polymorphisms (11) to (15), it is most preferable toanalyze all these polymorphisms, that is, five polymorphisms. In thecase where detection is carried out by combining four or less ofpolymorphisms, it is possible to preferentially select the polymorphismswith higher odds ratios in Examples mentioned below. For example, in thecase where four polymorphisms are used in combination, it is preferableto select four polymorphisms with higher odds ratio, that is, to select(11), (12), (14) and (15). Similarly, in the case where threepolymorphisms are used in combination, it is preferable to select (11),(12) and (15). Similarly, in the case where two polymorphisms are usedin combination, it is preferable to select (11) and (15).

A method for analyzing each genetic polymorphism is not particularlylimited. Known methods may include, for example, amplification by PCRusing an allele-specific primer (and probe), a method for polymorphismanalysis of amplified product by means of fluorescence or luminescence,PCR-RFLP (polymerase chain reaction-restriction fragment lengthpolymorphism) method, PCR-SSCP (polymerase chain reaction-single strandconformation polymorphism) method (Orita, M. et al., Proc. Natl. Acad.Sci., U.S.A., 86, 2766-2770 (1989), etc.), PCR-SSO (specific sequenceoligonucleotide) method, which use PCR method, ASO (allele specificoligonucleotide) hybridization method combining the PCR-SSO method and adot hybridization method (Saiki, Nature, 324, 163-166 (1986), etc.), orTaqMan-PCR method (Livak, K J, Genet Anal, 14, 143 (1999), Morris, T. etal., J. Clin. Microbiol. ,34, 2933 (1996)), Invader method (Lyamichev Vet al., Nat Biotechnol, 17, 292 (1999)), MALDI-TOF/MS (matrix) methodusing a primer extension method (Haff L A, Smirnov I P, Genome Res 7,378 (1997)), RCA (rolling cycle amplification) method (Lizardi P M etal., Nat Genet 19,225 (1998)), a method using DNA microchip ormicro-array (Wang D G et al., Science 280, 1077 (1998), etc.)), a primerextension method, a Southern blot hybridization method, a dothybridization method (Southern, E., J. Mol. Biol. 98, 503-517 (1975)),etc.), or the like. Furthermore, an analysis may be made by directsequencing of the portion of polymorphism which is subject to analysis.Note here that polymorphisms may be analyzed by combining these methodsad libitum.

In the case where the amount of nucleic acid sample is small, it ispreferable to analyze it by a method using PCR (for example, PCR-RFLPmethod) from the viewpoint of detection sensitivity or accuracy.Furthermore, any of the above-mentioned analysis methods may be employedafter nucleic acid sample is amplified in advance (including a partialregion of nucleic acid sample) by a gene amplification such as PCRmethod or a method applying PCR method.

Meanwhile, in the case where a large number of nucleic acid samples areanalyzed, a method capable of analyzing a large number of samples in arelatively short period of time, particularly, for example,allele-specific PCR method, allele-specific hybridization method,TaqMan-PCR method, Invader method, MALDI-TOF/MS (matrix) method usingprimary extension method, RCA (rolling cycle amplification) method, or amethod using a DNA chip or a micro-array.

The above methods use nucleic acids (also called “nucleic acids forpolymorphism analysis” in the present invention), e.g., primer and probein accordance with each method. Example of the nucleic acids forpolymorphism analysis may include: a nucleic acid with a sequencecomplementary to a given region including the site of polymorphism(partial DNA region) in the gene which contains polymorphism that issubject to the analysis; and a nucleic acid (primer) which has asequence complementary to a given region including the site ofpolymorphism(partial DNA region) and which is designed to allow thespecific amplification of the DNA fragment containing the relevant siteof polymorphism. In the case where polymorphism at position 1019 of theconnexin 37 gene is a subject to be analyzed, an example of such nucleicacids includes a nucleic acid having a sequence complementary to apartial DNA region including the position 1019 of the connexin 37 genein which the base at position 1019 is C (cytosine), or a nucleic acidhaving a sequence complementary to a partial DNA region including theposition 1019 of the connexin 37 gene in which the base at position 1019is T (thymine).

Other concrete examples of nucleic acids for polymorphism analysis mayinclude a set of nucleic acids which is designed to specifically amplifythe partial DNA region that contains the relevant site of polymorphismonly in the case where the site of polymorphism that is a subject to theanalysis is a certain genotype. A more concrete example may include forexample, a set of nucleic acids which is designed to specificallyamplify the partial DNA region including the site of polymorphism thatis subject to the analysis and which consists of a sense primer thatspecifically hybridizes the partial DNA region including the relevantsite of polymorphism in an antisense strand whose site of polymorphismis a certain genotype and of an antisense primer that specificallyhybridizes a partial region of the sense strand. In the case wherepolymorphism at position 1019 of the connexin 37 gene is a subject tothe analysis, examples of such a set of nucleic acids include a set ofnucleic acids which is designed to specifically amplify the partial DNAregion including the base at position 1019 of the connexin 37 gene andwhich consists of a sense primer that specifically hybridizes thepartial DNA region containing the base at position 1019 in the antisensestrand of the connexin 37 gene whose base at 1019 is C (cytosine) and ofan antisense primer that specifically hybridizes a partial region of thesense strand; or a set of nucleic acids which consists of a sense primerthat specifically hybridizes the partial DNA region including the baseat position 1019 in the antisense strand of the connexin 37 gene whosebase at position 1019 is T (thymine) and of an antisense primer thatspecifically hybridizes a partial region of the sense strand. The lengthof the partial DNA region to be amplified here is set accordingly in arange which is appropriate for its detection, and is for example, 50 bpto 200 bp, and preferably 80 bp to 150 bp. More concrete example of theset of nucleic acids for analyzing the connexin 37 (1019C→T)polymorphism for example may include a set containing the followingsequences. Note here that an underlined part in the following sequencesmeans a part corresponding to the polymorphism. Furthermore, in thesequence, N denotes any of A, T, C and G.

Sense primer CTCAGAATGGCCAAAANCC:, SEQ ID NO: 15 orCCTCAGAATGGCCAAAANTC: SEQ ID NO: 16 Antisense primer GCAGAGCTGCTGGGACGA:SEQ ID NO: 17

Similarly, an example of a nucleic acid primer for analyzing the TNFα(−863C→A) polymorphism may include a set of nucleic acids including thefollowing sequences.

Antisense primer GGCCCTGTCTTCGTTAANGG:, SEQ ID NO: 18 orATGGCCCTGTCTTCGTTAANTG: SEQ ID NO: 19 Sense primerCCAGGGCTATGGAAGTCGAGTATC: SEQ ID NO: 20

Similarly, an example of a nucleic acid primer for analyzing theNADH/NADPH oxidase p22 phox (242C→T) polymorphism may include a set ofnucleic acids including the following sequences.

Antisense primer ACCACGGCGGTCATGNGC:, SEQ ID NO: 21 orACCACGGCGGTCATGNAC: SEQ ID NO: 22 Sense primer GCAGCAAAGGAGTCCCGAGT: SEQID NO: 23

Similarly, an example of a nucleic acid primer for analyzing theangiotensinogen (−6G→A) polymorphism may include a set of nucleic acidsincluding the following sequences.

Antisense primer CGGCAGCTTCTTCCCNCG:. SEQ ID NO: 24 orCGGCAGCTTCTTCCCNTG: SEQ ID NO: 25 Sense primer CCACCCCTCAGCTATAAATAGG:SEQ ID NO: 26

Similarly, an example of a nucleic acid primer for analyzing the Apo E(−219G→T) polymorphism may include a set of nucleic acids including thefollowing sequences.

Sense primer GAATGGAGGAGGGTGTCTNGA:, SEQ ID NO: 27 orAGAATGGAGGAGGGTGTCTNTA: SEQ ID NO: 28 Antisense primerCCAGGAAGGGAGGACACCTC: SEQ ID NO: 29

Similarly, an example of a nucleic acid primer for analyzing the PAFacetylhydrolase (994G→T) polymorphism may include a set of nucleic acidsincluding the following sequences.

Sense primer TTCTTTTGGTGGAGCAACNGT:, SEQ ID NO: 30 orATTCTTTTGGTGGAGCAACNTT: SEQ ID NO: 31 Antisense primerTCTTACCTGAATCTCTGATCTTCA: SEQ ID NO: 32

Similarly, an example of a nucleic acid primer for analyzing the ApoC-III (−482C→T) polymorphism may include a set of nucleic acidsincluding the following sequences.

Sense primer CGGAGCCACTGATGCNCG:, SEQ ID NO: 33 or CGGAGCCACTGATGCNTG:SEQ ID NO: 34 Antisense primer TGTTTGGAGTAAAGGCACAGAA: SEQ ID NO: 35

Similarly, an example of a nucleic acid primer for analyzing the TSP4(1186G→C) polymorphism may include a set of nucleic acids including thefollowing sequences.

Sense primer CGAGTTGGGAACGCACNCT:, SEQ ID NO: 36 or CGAGTTGGGAACGCACNGT:SEQ ID NO: 37 Antisense primer GGTCTGCACTGACATTGATGAG: SEQ ID NO: 38

Similarly, an example of a nucleic acid primer for analyzing theIL-10(−819T→C) polymorphism may include a set of nucleic acids includingthe following sequences.

Sense primer TACCCTTGTACAGGTGATGTANTA:. SEQ ID NO: 39 orTACCCTTGTACAGGTGATGTANCA: SEQ ID NO: 40 Antisense primerATAGTGAGCAAACTGAGGCACA: SEQ ID NO: 41

Similarly, an example of a nucleic acid primer for analyzing the IL-10(−592A→C) polymorphism may include a set including the followingsequences.

Antisense primer CAGAGACTGGCTTCCTACANGA:, SEQ ID NO: 42 orCCAGAGACTGGCTTCCTACANTA: SEQ ID NO: 43 Sense primerGCCTGGAACACATCCTGTGA: SEQ ID NO: 44

Similarly, an example of a nucleic acid primer for analyzing thestromelysin 1 (−1171/5A→6A) polymorphism may include a set of nucleicacids including the following sequences.

Sense primer TTTGATGGGGGGAAAANAC:, SEQ ID NO: 45 or TTGATGGGGGGAAAANCC:SEQ ID NO: 46 Antisense primer CCTCATATCAATGTGGCCAA: SEQ ID NO: 47

Similarly, an example of a nucleic acid primer for analyzing the PAI1(−668/4G→5G) polymorphism may include a set of nucleic acids includingthe following sequences.

Sense primer GGCACAGAGAGAGTCTGGACACG: SEQ ID NO: 48 Antisense primerGGCCGCCTCCGATGATACA: SEQ ID NO: 49

Similarly, an example of a nucleic acid primer for analyzing theglycoprotein Ibα (1018C→T) polymorphism may include a set of nucleicacids including the following sequences.

Sense primer CCCAGGGCTCCTGNCG:, SEQ ID NO: 50 or CCCCAGGGCTCCTGNTG: SEQID NO: 51 Antisense primer TGAGCTTCTCCAGCTTGGGTG: SEQ ID NO: 52

Similarly, an example of a nucleic acid primer for analyzing theparaoxonase (584G→A) polymorphism may include a set of nucleic acidsincluding the following sequences.

Sense primer ACCCAAATACATCTCCCAGGANCG:, SEQ ID NO: 53 orAACCCAAATACATCTCCCAGGNCT: SEQ ID NO: 54 Antisense primerGAATGATATTGTTGCTGTGGGAC: SEQ ID NO: 55

Similarly, an example of a nucleic acid primer for analyzing the Apo E(4070C→T) polymorphism may include a set of nucleic acids including thefollowing sequences.

Sense primer CCGATGACCTGCAGAANCG:, SEQ ID NO: 56 orGCCGATGACCTGCAGAANTG: SEQ ID NO: 57 Antisense primerCGGCCTGGTACACTGCCAG: SEQ ID NO: 58

On the other hand, a concrete example of the probe can include:

as a probe for analyzing Apo C-III (−482C→T) polymorphism,

AGCCACTGATGCNCGGTCT:, SEQ ID NO: 59 or AGCCACTGATGCNTGGTCT:, SEQ ID NO:60as a probe for analyzing IL-10 (−819T→C) polymorphism,

GTACAGGTGATGTANTATCTCTGTG:, SEQ ID NO: 61 or,GTACAGGTGATGTANCATCTCTGTG:, SEQ ID NO: 62 andas a probe for analyzing PAI1 (−668/4G→5G) polymorphism,

TGGACACGTGGGGGAGTCAG:, SEQ ID NO: 63 or TGGACACGTGGGGAGTCAGC:. SEQ IDNO: 64

The above nucleic acid primers and nucleic acid probes are mereexamples. Nucleic acid primers may contain a partially modified basesequence as long as they can carry out the aimed amplification reactionwithout inconvenience, while nucleic acid probes may contain a partiallymodified base sequence as long as they can carry out the aimedhybridization reaction without inconvenience. “Partially modified”herein means that part of bases is deleted, replaced, inserted, and/oradded. The numbers of modified bases are for example one to seven,preferably one to five, and more preferably one to three. Note here thatsuch a modification is made in the portions other than bases whichcorrespond to the site of polymorphism, in principle. However, in thecase where the polymorphism that is a subject of analysis is stromelysin1 (−1171/5A→6A) polymorphism or PAI1 (−668/4G→5G) polymorphism, primersor probes obtained by modifying a part of base corresponding to apolymorphism site may be used.

As nucleic acids for polymorphism analysis (probes or primers), DNAfragments or RNA fragments are used accordingly in response to theanalysis method employed. The base length of nucleic acids forpolymorphism analysis may be sufficient if it exerts respectivefunctions of each nucleic acid. Base lengths in the case of use asprimers are for example, 10 to 50 bp, preferably 15 to 40 bp, and morepreferably 15 to 30 bp.

Note here that in the case of use as primers, some mismatches to thesequence which constitutes the template may be admitted as long as theprimer can specifically hybridize the subject for amplification andamplify the target DNA fragment. In the case of probes, some mismatchesto the sequence which is subject to detection may be similarly admittedas long as the probe can specifically hybridize the sequence which issubject to detection. The numbers of mismatches are one to several,preferably one to five, and more preferably one to three.

Nucleic acids for polymorphism analysis (primers and probes) can besynthesized in accordance with known methods, e.g., phosphodiestermethod. Note here that textbooks (e.g., Molecular Cloning, ThirdEdition, Cold Spring Harbor Laboratory Press, New York) can be referredwith respect to the design, synthesis, and others of nucleic acids forpolymorphism analysis.

Nucleic acids for polymorphism analysis in the present invention can belabeled with labeling substances in advance. The use of such labelednucleic acids allows, for example, the analysis of polymorphism by usingthe labeling amount in the product of amplification as a marker.Furthermore, by labeling two kinds of primers which were designedspecifically amplify the partial DNA region in the gene of each genotypethat constitute polymorphism with labeling substances that are differentfrom each other, the genotype of a nucleic acid sample can bediscriminated according to the labeling substance and labeling amount tobe detected based on the product of amplification. Concrete examples ofdetection methods using these labeled primers may include: a method fordetecting polymorphism, comprising labeling, with fluorescein isocyanateand Texas red, two kinds of nucleic acid primers (allele-specific senseprimers) that respectively and specifically hybridize the sense strandof each genotype constituting polymorphism; amplifying the partial DNAregion including the site of polymorphism by using these labeled primersand the antisense primers that specifically hybridize the antisensestrand; and measuring the labeling amount of each fluorescent substancein the product of amplification obtained. Note here that labeling of theantisense primer herein with for example, biotin allows the separationof the product of amplification by utilizing the specific bindingbetween biotin and avidin.

Radioactive isotopes, for example, ³²P, and fluorescent substance, forexample, fluorescein isothiocyanate, tetramethyl rhodamineisothiocyanate, and Texas red, etc. can be exemplified as labelingsubstances to be used in labeling nucleic acids for polymorphismanalysis. The 5′ terminal labeling method using alkaline phosphatase andT4 polynucleotide kinase, the 3′ terminal labeling method using T4 DNApolymerase and Klenow fragment, nicktranslation method, random primermethod (Molecular Cloning, Third Edition, Chapter 9, Cold Spring HarborLaboratory Press, New York), and the like can be exemplified as labelingmethods.

The above-mentioned nucleic acids for polymorphism analysis can be usedalso under a condition fixed to an insoluble support. Processing of aninsoluble support to be used for the fixation to several forms such aschips and beads allows the more simplified analysis of polymorphism byusing these fixed nucleic acids.

A nucleic acid sample can be prepared from blood, skin cells, mucouscells, hair, and others from the subject according to known extractionmethods and purification methods. In the case of including the genewhich is subject to the analysis of polymorphism, the genome DNA ofarbitrary length can be used as a nucleic acid sample. Furthermore, itis not necessary to use a nucleic acid sample in which all genes subjectto the analysis are present on one nucleic acid. That is to say, as anucleic acid sample of the present invention, both material in which allgenes subject to the analysis are present on one nucleic acid andmaterial in which genes subject to the analysis are present separatelyon two or more nucleic acids can be used. Note here that material in afragmented or partial condition may be accepted as long as the site ofpolymorphism to be analyzed is at least present, although genes subjectto the analysis in a nucleic acid sample are not in a complete condition(i.e., a condition in which the full length of the gene is present).

Analysis of each gene polymorphism is carried out each by each of thegene polymorphism or a plurality or entire gene polymorphisms arecarried out simultaneously. In the former case, for example, nucleicacid sample collected from the subjects is divided in accordance withthe number of polymorphisms to be analyzed, and analysis of polymorphismis carried out individually. In the latter case, for example, analysisof polymorphism can be carried out by DNA chip or micro-array. Note herethat “simultaneousness” herein not only imply that all operations of theanalysis process are conducted simultaneously but also include the casein which part of operations (e.g., operation to amplify nucleic acid,hybridization or detection of the probe) is conducted simultaneously.

Polymorphism of each gene can be analyzed by using mRNA which is aproduct of transcription of the gene which is subject to the analysis.After extracting and purifying mRNA of the gene which is subject to theanalysis from blood, urine, and others of the subject, for example,polymorphism can be analyzed with mRNA as a starting material byconducting methods, e.g., Northern blotting method (Molecular Cloning,Third Edition, 7.42, Cold Spring Harbor Laboratory Press, New York), dotblotting method (Molecular Cloning, Third Edition, 7.46, Cold SpringHarbor Laboratory Press, New York), RT-PCR method (Molecular Cloning,Third Edition, 8.46, Cold Spring Harbor Laboratory Press, New York), andmethods using the DNA chip (DNA array), and the like.

In addition, in the above-mentioned polymorphism, polymorphism involvedwith changes in amino acids can analyzed by using the expression productof gene that is a subject to analysis. In this case, material, evenbeing partial protein or partial peptide, can be used as a sample foranalysis as long as it contains amino acids which correspond to the siteof polymorphism.

Analysis methods using these expression products of gene may include: amethod for directly analyzing amino acids at the site of polymorphism, amethod for immunologically analyzing utilizing changes ofthree-dimensional structure, or the like. As the former, a well-knownamino acid sequence analysis method (a method using Edman method) can beused. As the latter, ELISA (enzyme-linked immunosorbent assay) usingthemonoclonal antibody or polyclonal antibody which has binding activityspecific to the expression product of gene which has any of genotypesthat constitute polymorphism; radioimmunoassay, immunoprecipitationmethod, immunodiffusion method, and the like can be used.

Information about polymorphism to be obtained by conducting thedetection methods of the present invention described above can be usedto diagnose a genetic risk of myocardial infarction. That is to say, thepresent invention also provides a method for diagnosing a genetic riskof myocardial infarction, which comprises a step for determining thegenotype in a nucleic acid sample based on information aboutpolymorphism that was obtained by the above-detection methods, and astep for assessing a genetic risk of myocardial infarction based on thedetermined genotype of the nucleic acid sample. Herein, thedetermination of the genotype is typically to determine which genotypeboth alleles of nucleic acid samples have with respect to thepolymorphism to be detected. In the case where the subject to bedetected is connexin 37 (1019C→T) polymorphism, for example, typically,investigation on what type the connexin 37 gene in a nucleic acid sampleis TT (the base at position 1019 is a homozygote of allele T), CT (thebase at position 1019 is a heterozygote of allele C and allele T) and CC(the base at position 1019 is a homozygote of allele C) in a nucleicacid sample the connexin 37 gene is.

By considering the results obtained in Example mentioned below, in orderto enable a diagnosis of genetic risk of myocardial infarction with highaccuracy and high predictability, for example, in the case of theconnexin 37 (1019C→T) polymorphism, it is determined whether thegenotype in a nucleic acid sample is TT or CT, or CC. Similarly, in thecase of the TNFα (−863C→A) polymorphism, it is determined whether thegenotype is AA or CA, or CC; in the case of NADH/NADPH oxidase p22 phox(242C→T) polymorphism, it is determined whether the genotype is TT orCT, or CC; in the case of angiotensinogen (−6G→A) polymorphism, it isdetermined whether the genotype is AA, or GA or GG; in the case of ApoE-219 (−219G→T) polymorphism, it is determined whether the genotype isTT, or GT or GG; in the case of PAF acetylhydrolase (994G→T)polymorphism, it is determined whether the genotype is TT or GT, or GG;in the case of Apo C-III (−482C→T) polymorphism, it is determinedwhether the genotype is TT, or CT or CC; in the case of TSP4 (1186G→C)polymorphism, it is determined whether the genotype is CC or GC, or GG;in the case of IL-10 (−819T→C) polymorphism, it is determined whetherthe genotype is CC, or CT or TT; in the case of IL-10 (−592A→C)polymorphism, it is determined whether the genotype is CC, or CA or AA;in the case of stromelysin 1 (−1171/5A→6A) polymorphism, it isdetermined whether the genotype is 6A/6A or 5A/6A, or 5A/5A; in the caseof PAI1 (−668/4G→5G) polymorphism, it is determined whether the genotypeis 5G/5G or 4G/5G, or 4G/4G; in the case of glycoprotein Ibα (1018C→T)polymorphism, it is determined whether the genotype is TT, or CT or CC;in the case of paraoxonase (584G→A) polymorphism, it is determinedwhether the genotype is AA, or GA or GG; and in the case of Apo E(4070C→T) polymorphism, it is determined whether the genotype is TT, orCT or CC.

Diagnosis of a genetic risk of myocardial infarction enables predictionof potentiality in that myocardial infraction might be developed in thefuture (susceptibility to development), that is, risk of development(predisposition to development). Furthermore, based on the objectiveindicator that is the genotype, it is possible to recognize myocardialinfarction and to evaluate the state of disease. In other words,according to the diagnosing method of the present invention, it ispossible to asses the risk of development of myocardial infarction or toevaluate the state of disease. Above all, it is extremely significantfrom the clinical viewpoint to carry out the assessment of the risk ofdevelopment. It is advantageous because awareness in advance of the riskof development contributes to primary prevention of myocardialinfarction and makes it possible to take an appropriate measurement.

Information to be obtained by a diagnostic method of the presentinvention can be used for selecting an appropriate treatment,improvement of prognosis, improvement in QOL (quality of life ofpatients), or reduction in risk of development.

By carrying out the diagnostic method of the present inventionregularly, it is possible to monitor, for example, the risk ofdevelopment of myocardial infarction. As a result of such monitoring, ifthe correlation between some external factors (environmental factors,administration of drugs, etc.) and the increase in the risk ofdevelopment is found, such external factors are recognized as riskfactors and it is thought that the risk of development can be reducedbased on this information.

By utilizing the genetic information associated with the development ofmyocardial infarction obtained by the present invention, it is possibleto carry out a treatment (including a preventive treatment) formyocardial infarction. For example, as a result of carrying out thediagnostic method of the present invention, in the case where thepolymorphism to be analyzed is a genotype to increase the risk ofdevelopment of myocardial infarction, by introducing and expressing agene having a genotype with low risk of development is introduced into aliving body, the reduction of disease, suppression of development, andreduction of risk of development can be expected due to the expressionof genes. The same treatment effect can be expected by a methodincluding: introducing antisense strand with respect to mRNA of genehaving a genotype with high risk of development and suppressing theexpression of the mRNA.

The introduction of gene or antisense strand can be carried out by amethod, for example, a method using a plasmid for gene introduction or avirus vector, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci.U.S.A. 81, 7161-7165(1984), an ultrasonic microbubble (Lawrie, A., etal. Gene Therapy 7, 2023-2027 (2000)), lipofection (Felgner, P. L. etal., Proc. Natl. Acad. Sci. U.S.A. 84, 7413-7417 (1984)), microinjection(Graessmann, M. & Graessmann, A., Proc. Natl. Acad. Sci. U.S.A.73,366-370(1976)), and the like. By utilizing these methods, desiredgenes, etc. can be directly introduced (in vivo method) or indirectlyintroduced (ex vivo method).

The second aspect of the present invention provides kits to be used inthe above-mentioned detecting method or diagnostic method in the presentinvention (kits for detecting the genotype or kits for diagnosingmyocardial infarction). Such kits contain nucleic acids (nucleic acidfor polymorphism analysis) for analyzing two or more polymorphismsselected from the group consisting of polymorphisms described in (1) to(10) above; or such kits contain nucleic acids (nucleic acid forpolymorphism analysis) for analyzing two or more polymorphisms selectedfrom the group consisting of polymorphisms described in (11) to (15)above. As further embodiment, kits are constructed, which containsnucleic acid for polymorphism analysis described in (15) above.

In the analysis methods by which it is applied (a method which utilizesPCR using the above-mentioned allele-specific nucleic acids and thelike, PCR-RFLP method, PCR-SSCP method, TaqMan-PCR method, Invadermethod, etc.), nucleic acids for polymorphism analysis are designed asmaterials which can specifically amplifies (primer) or specificallydetect (probe) the DNA region containing the polymorphism portion to beanalyzed or mRNA which corresponds to the region. Concrete examples ofkits to be provided according to the present invention are describedbelow.

A kit for detecting the genotype, comprising two or more nucleic acidsselected from the group consisting of the below-mentioned (1) to (10):

-   -   (1) a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position 1019 of        the connexin 37 gene whose base at position 1019 is C, or a        nucleic-acid having a sequence which is complementary to the        partial DNA region containing the base at position 1019 of the        connexin 37 gene whose base at position 1019 is T:    -   (2) a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position −863 of        the tumor necrosis factor α gene whose base at position −863 is        C, or a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position −863 of        the tumor necrosis factor α gene whose base at position −863 is        A:    -   (3) a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position 242 of        the NADH/NADPH oxidase p22 phox gene whose base at position 242        is C, or a nucleic acid having a sequence which is complementary        to the partial DNA region containing the base at position 242 of        the NADH/NADPH oxidase p22 phox gene whose base at position 242        is T:    -   (4) a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position −6 of the        angiotensinogen gene whose base at position −6 is G, or a        nucleic acid having a sequence which is complementary to the        partial DNA region containing the base at position −6 of the        angiotensinogen gene whose base at position −6 is A:    -   (5) a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position −219 of        the apolipoprotein E gene whose base at position −219 is G, or a        nucleic acid having a sequence which is complementary to the        partial DNA region containing the base at position −219 of the        apolipoprotein E gene whose base at position −219 is T:    -   (6) a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position 994 of        the platelet-activating factor acetylhydrolase gene whose base        at position 994 is G, or a nucleic acid having a sequence which        is complementary to the partial DNA region containing the base        at position 994 of the platelet-activating factor        acetylhydrolase gene whose base at position 994 is T:    -   (7) a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position −482 of        the apolipoprotein C-III gene whose base at position −482 is C,        or a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position −482 of        the apolipoprotein C-III gene whose base at position −482 is T:    -   (8) a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position 1186 of        the thrombospondin 4 gene whose base at position 1186 is G, or a        nucleic acid having a sequence which is complementary to the        partial DNA region containing the base at position 1186 of the        thrombospondin 4 gene whose base at position 1186 is C:    -   (9) a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position −819 of        the interleukin-10 gene whose base at position −819 is T, or a        nucleic acid having a sequence which is complementary to the        partial DNA region containing the base at position −819 of the        interleukin-10 gene whose base at position −819 is C: and    -   (10) a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position −592 of        the interleukin-10 gene whose base at position −592 is A, or a        nucleic acid having a sequence which is complementary to the        partial DNA region containing the base at position −592 of the        interleukin-10 gene whose base at position −592 is C.

In the above mention, kits are constructed by selecting two or morenucleic acids from the group consisting of (1) to (10). However, kitsmay be constructed by making a group consisting of two or more nucleicacids arbitrarily selected from (1) to (10) and selecting two or morenucleic acids from such a group. For example, kits are constructed byselecting two or more nucleic acids from the group consisting of (1),(5), (6), (8), (9) and (10) (nucleic acids for polymorphism analysiswith odds ratio of one or more in Example mentioned below), or kits areconstructed by selecting two or more nucleic acids from the groupconsisting of (1), (5), (6), (8) and (9) (nucleic acids for polymorphismanalysis with five highest odds ratios in Example mentioned below).

A kit for detecting the genotype, comprising two or more nucleic acidsselected from the group consisting of the following (11) to (15):

-   -   (11) a nucleic acid having a sequence which is complementarily        to the partial DNA region containing the part of sequence of the        stromelysin 1 gene in which five A successively exist in the 3′        direction from the position −1171, or a nucleic acid having a        sequence which is complementary to the partial DNA region        containing the part of sequence of the stromelysin 1 gene in        which six A successively exist in the 3′ direction from the        position −1171;    -   (12) a nucleic acid having a sequence which is complementary to        the partial DNA region containing the part of sequence of the        plasminogen activator inhibitor 1 gene in which four G        successively exist in the 3′ direction from the position −668,        or a nucleic acid having a sequence which is complementary to        the partial DNA region containing the part of sequence of the        plasminogen activator inhibitor 1 gene in which five G        successively exist in the 3′ direction from the position −668 in        the 3′ direction;    -   (13) a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position 1018 of        the glycoprotein Ibα gene whose base at position 1018 is C, or a        nucleic acid having a sequence which is complementary to the        partial DNA region containing the base at position 1018 of the        glycoprotein Ibα gene whose base at position 1018 is T;    -   (14) a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position 584 of        the paraoxonase gene whose base at position 584 is G, or a        nucleic acid having a sequence which is complementary to the        partial DNA region containing the base at position 584 of the        paraoxonase gene whose base at position 584 is A; and    -   (15) a nucleic acid having a sequence which is complementary to        the partial DNA region containing the base at position 4070 of        the apolipoprotein E gene whose base at position 4070 is C, or a        nucleic acid having a sequence which is complementary to the        partial DNA region containing the base at position 4070 of the        apolipoprotein E gene whose base at position 4070 is T.

In the above mention, kits are constructed by selecting two or morenucleic acids from the group consisting of (11) to (15). However, kitsmay be constructed by making a group consisting of two or more nucleicacids arbitrarily selected from (11) to (15) and selecting two or morenucleic acids from such a group. For example, kits are constructed byselecting two or more nucleic acids from the group consisting of (11),(12), (14) and (15) (nucleic acids for polymorphism analysis with oddsratio of one or more in Example mentioned below), or kits areconstructed by selecting two or more nucleic acids from the groupconsisting of (11), (12) and (15) (nucleic-acids for polymorphismanalysis with three highest odds ratios in Example mentioned below).

A kit for detecting the genotype comprising the following nucleic acid:

a nucleic acid having a sequence which is complementary to the partialDNA region containing the base at position 4070 of the apolipoprotein Egene whose base at position 4070 is C, or a nucleic acid having asequence which is complementary to the partial DNA region containing thebase at position 4070 of the apolipoprotein E gene whose base atposition 4070 is T.

A kit for detecting the genotype, comprising two or more sets of nucleicacids selected from the group consisting of the following (1) to (10):

-   -   (1) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        1019 of the connexin 37 gene only in the case where the base at        position 1019 of the connexin 37 gene in a nucleic acid sample        is C, or a set of nucleic acids which is designed to        specifically amplify the partial DNA region containing the base        at position 1019 of the connexin 37 gene only in the case where        the base at position 1019 of the connexin 37 gene in a nucleic        acid sample is T;    -   (2) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        −863 of the tumor necrosis factor α gene only in the case where        the base at position −863 of the tumor necrosis factor α gene in        a nucleic acid sample is C, or a set of nucleic acids which is        designed to specifically amplify the partial DNA region        containing the base at position −863 of the tumor necrosis        factor α gene only in the case where the base at position −863        of the tumor necrosis factor α gene in a nucleic acid sample is        A;    -   (3) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        242 of the NADH/NADPH oxidase p22 phox gene only in the case        where the base at position 242 of the NADH/NADPH oxidase p22        phox gene in a nucleic acid sample is C, or a set of nucleic        acids which is designed to specifically amplify the partial DNA        region containing the base at position 242 of the NADH/NADPH        oxidase p22 phox gene only in the case where the base at        position 242 of the NADH/NADPH oxidase p22 phox gene in a        nucleic acid sample is T;    -   (4) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        −6 of the angiotensinogen gene only in the case where the base        at position −6 of the angiotensinogen gene in a nucleic acid        sample is G, or a set of nucleic acids which is designed to        specifically amplify the partial DNA region containing the base        at position −6 of the angiotensinogen gene only in the case        where the base at position −6 of the angiotensinogen gene in a        nucleic acid sample is A;    -   (5) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        −219 of the apolipoprotein E gene only in the case where the        base at position −219 of the apolipoprotein E gene in a nucleic        acid sample is G, or a set of nucleic acids which is designed to        specifically amplify the partial DNA region containing the base        at position −219 of the apolipoprotein E gene only in the case        where the base at position −219 of the apolipoprotein E gene in        a nucleic acid sample is T;    -   (6) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        994 of the platelet-activating factor acetylhydrolase gene only        in the case where the base at position 994 of the        platelet-activating factor acetylhydrolase gene in a nucleic        acid sample is G, or a set of nucleic acids which is designed to        specifically amplify the partial DNA region containing the base        at position 994 of the platelet-activating factor        acetylhydrolase gene only in the case where the base at position        994 of the platelet-activating factor acetylhydrolase gene in a        nucleic acid sample is T;    -   (7) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        −482 of the apolipoprotein C-III gene only in the case where the        base at position −482 of the apolipoprotein C-III gene in a        nucleic acid sample is C, or a set of nucleic acids which is        designed to specifically amplify the partial DNA region        containing the base at position −482 of the apolipoprotein C-III        gene only in the case where the base at position −482 of the        apolipoprotein C-III gene in a nucleic acid sample is T;    -   (8) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        1186 of the thrombospondin 4 gene only in the case where the        base at position 1186 of the thrombospondin 4 gene in a nucleic        acid sample is G, or a set of nucleic acids which is designed to        specifically amplify the partial DNA region containing the base        at position 1186 of the thrombospondin 4 gene only in the case        where the base at position 1186 of the thrombospondin 4 gene in        a nucleic acid sample is C;    -   (9) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        −819 of the interleukin-10 gene only in the case where the base        at position −819 of the interleukin-10 gene in a nucleic acid        sample is T, or a set of nucleic acids which is designed to        specifically amplify the partial DNA region containing the base        at position −819 of the interleukin-10 gene only in the case        where the base at position −819 of the interleukin-10 gene in a        nucleic acid sample is C; and    -   (10) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        −592 of the interleukin-10 gene only in the case where the base        at position −592of the interleukin-10 gene in a nucleic acid        sample is A, or a set of nucleic acids which, is designed to        specifically amplify the partial DNA region containing the base        at position −592 of the interleukin-10 gene only in the case        where the base at position −592 of the interleukin-10 gene in a        nucleic acid sample is C;

In the above mention, kits are constructed by selecting two or more setsof nucleic acids from the group consisting of (1) to (10). However, kitsmay be constructed by making a group consisting of two or more sets ofnucleic acids arbitrarily selected from (1) to (10) and selecting two ormore sets of nucleic acids from such a group. For example, kits areconstructed by selecting two or more sets of nucleic acids from thegroup consisting of (1), (5), (6), (8), (9) and (10) (nucleic acids forpolymorphism analysis with odds ratio of one or more in Examplementioned below), or kits are constructed by selecting two or more setsof nucleic acids from the group consisting of (1), (5), (6), (8) and (9)(nucleic acids for polymorphism analysis with five highest odds ratiosin Example mentioned below).

A kit for detecting the genotype, comprising two or more sets of nucleicacids selected from the group consisting of the following (11) to (15):

-   -   (11) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the part of sequence        of the stromelysin 1 gene only in the case where five A exist        successively in the 3′ direction from the position −1171 in the        stromelysin 1 gene in a nucleic acid sample, or a set of nucleic        acids which is designed to specifically amplify the partial DNA        region containing the part of sequence of the stromelysin 1 gene        only in the case where six A exist successively in the 3′        direction from the position −1171 in the stromelysin 1 gene in a        nucleic acid sample;    -   (12) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the part of sequence        of the plasminogen activator inhibitor 1 gene only in the case        where four G exist successively in the 3′ direction from the        position −668 in the plasminogen activator inhibitor 1 gene in a        nucleic acid sample, or a set of nucleic acids which is designed        to specifically amplify the partial DNA region containing the        part of sequence of the plasminogen activator inhibitor 1 gene        only in the case where five G exist successively in the 3′        direction from the position −668 in the plasminogen activator        inhibitor 1 gene in a nucleic acid sample;    -   (13) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        1018 of the glycoprotein Ibα gene only in the case where the        base at position 1018 of the glycoprotein Ibα gene in a nucleic        acid sample is C, or a set of nucleic acids which is designed to        specifically amplify the partial DNA region containing the base        at position 1018 of glycoprotein Ibα gene only in the case where        the base at position 1018 of the glycoprotein Ibα gene in a        nucleic acid sample is T;    -   (14) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        584 of the paraoxonase gene only in the case where the base at        position 584 of the paraoxonase gene in a nucleic acid sample is        G, or a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        584 of the paraoxonase gene only in the case where the base at        position 584 of paraoxonase gene in a nucleic acid sample is A;        and    -   (15) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        4070 of the apolipoprotein E gene only in the case where the        base at position 4070 of the apolipoprotein E gene in a nucleic        acid sample is C, or a set of nucleic acids which is designed to        specifically amplify the partial DNA region containing the base        at position 4070 of the apolipoprotein E gene only in the case        where the base at position 4070 of the apolipoprotein E gene in        a nucleic acid sample is T.

In the above mention, kits are constructed by selecting two or more setsof nucleic acids from the group consisting of (11) to (15). However,kits may be constructed by making a group consisting of two or more setsof nucleic acids arbitrarily selected from (11) to (15) and selectingtwo or more sets of nucleic acids from such a group. For example, kitsmay be constructed by selecting two or more sets of nucleic acids fromthe group consisting of (11), (12), (14) and (15) (sets of nucleic acidsfor polymorphism analysis with odds ratio of one or more in Examplementioned below), or kits may be constructed by selecting two or moresets of nucleic acids from the group consisting of (11), (12) and (15)(sets of nucleic acid for polymorphism analysis with three highest oddsratios in Example mentioned below).

A kit for detecting the genotype, comprising the following set ofnucleic acids:

a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 4070 of theapolipoprotein E gene only in the case where the base at position 4070of the apolipoprotein E gene in a nucleic acid sample is C, or a set ofnucleic acids which is designed to specifically amplify the partial DNAregion containing the base at position 4070 of the apolipoprotein E geneonly in the case where the base at position 4070 of the apolipoprotein Egene in a nucleic acid sample is T.

A kit for detecting the genotype, comprising two or more sets of nucleicacids selected from the group consisting of the following (1) to (10):

-   -   (1) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        1019 of the connexin 37 gene and which consists of a sense        primer that specifically hybridizes the partial DNA region        containing the base at position 1019 of the connexin 37 gene        whose base at position 1019 is C and/or a sense primer that        specifically hybridizes the partial DNA region containing the        base at position 1019 in the connexin 37 gene whose gene at        position 1019 is T and of an antisense primer that specifically        hybridizes a partial portion of the connexin 37 gene;    -   (2) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        −863 of the tumor necrosis factor α gene and which consists of        an antisense primer that specifically hybridizes the partial DNA        region containing the base at position −863 of the tumor        necrosis factor α gene whose base at position −863 is C and/or        an antisense primer that specifically hybridizes the partial DNA        region containing the base at position −863 in the tumor        necrosis factor a gene whose gene at position −863 is A and of a        sense primer that specifically hybridizes a partial portion of        the tumor necrosis factor α gene;    -   (3) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        242 of the NADH/NADPH oxidase p22 phox gene and which consists        of an antisense primer that specifically hybridizes the partial        DNA region containing the base at position −863 of the        NADH/NADPH oxidase p22 phox gene whose base at position 242 is C        and/or an antisense primer that specifically hybridizes the        partial DNA region containing the base at position 242 in the        NADH/NADPH oxidase p22 phox gene whose gene at position 242 is T        and of a sense primer that specifically hybridizes a partial        portion of the NADH/NADPH oxidase p22 phox gene;    -   (4) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        −6 of the angiotensinogen gene and which consists of an        antisense primer that specifically hybridizes the partial DNA        region containing the base at position −6 of the angiotensinogen        gene whose base at position −6 is G and/or an antisense primer        that specifically hybridizes the partial DNA region containing        the base at position −6 in the angiotensinogen gene whose gene        at position −6 is A and of a sense primer that specifically        hybridizes a partial portion of the angiotensinogen gene;    -   (5) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        −219 of the apolipoprotein E gene and which consists of a sense        primer that specifically hybridizes the partial DNA region        containing the base at position −219 of the apolipoprotein E        gene whose base at position −219 is G and/or a sense primer that        specifically hybridizes the partial DNA region containing the        base at position −219 in the apolipoprotein E gene whose gene at        position −219 is T and of an antisense primer that specifically        hybridizes a partial portion of the apolipoprotein E gene;    -   (6) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        994 of the platelet-activating factor acetylhydrolase gene and        which consists of a sense primer that specifically hybridizes        the partial DNA region containing the base at position 994 of        the platelet-activating factor acetylhydrolase gene whose base        at position 994 is G and/or a sense primer that specifically        hybridizes the partial DNA region containing the base at        position 994 in the platelet-activating factor acetylhydrolase        gene whose gene at position 994 is T and of an antisense primer        that specifically hybridizes a partial portion of the        platelet-activating factor acetylhydrolase gene;    -   (7) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        −482 of the apolipoprotein C-III gene and which consists of a        sense primer that specifically hybridizes the partial DNA region        containing the base at position −482 of the apolipoprotein C-III        gene whose base at position −482 is C and/or a sense primer that        specifically hybridizes the partial DNA region containing the        base at position −482 in the apolipoprotein C-III gene whose        gene at position −482 is T and of an antisense primer that        specifically hybridizes a partial portion of the apolipoprotein        C-III gene;    -   (8) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        1186 of the thrombospondin 4 gene and which consists of a sense        primer that specifically hybridizes the partial DNA region        containing the base at position 1186 of the thrombospondin 4        whose base at position 1186 is G and/or a sense primer that        specifically hybridizes the partial DNA region containing the        base at position 1186 in the thrombospondin 4 gene whose gene at        position 1186 is C and of an antisense primer that specifically        hybridizes a partial portion of the thrombospondin 4 gene;    -   (9) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        −819 of the interleukin-10 gene and which consists of a sense        primer that specifically hybridizes the partial DNA region        containing the base at position −819 of the interleukin-10 whose        base at position −819 is T and/or a sense primer that        specifically hybridizes the partial DNA region containing the        base at position −819 in the interleukin-10 gene whose gene at        position −1186 is C and of an antisense primer that specifically        hybridizes a partial portion of the interleukin-10 gene; and    -   (10) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        −592 of the interleukin-10 gene and which consists of an        antisense primer that specifically hybridizes the partial DNA        region containing the base at position −592 of the        interleukin-10 whose base at position −592 is A and/or an        antisense primer that specifically hybridizes the partial DNA        region containing the base at position −592 in the        interleukin-10 gene whose gene at position −592 is C and of a        sense primer that specifically hybridizes a partial portion of        the interleukin-10 gene.

In the above mention, kits are constructed by selecting two or more setsof nucleic acids from the group consisting of (1) to (10). However, kitsmay be constructed by making a group consisting of two or more sets ofnucleic acids arbitrarily selected from (1) to (10) and selecting two ormore sets of nucleic acids from such a group. For example, kits areconstructed by selecting two or more nucleic acids from the groupconsisting of (1), (5), (6), (8), (9) and (10) (sets of nucleic acidsfor polymorphism analysis with odds ratio of one or more in Examplementioned below), or kits are constructed by selecting two or more setsof nucleic acids from the group consisting of (1), (5), (6), (8) and (9)(sets of nucleic acids for polymorphism analysis with five highest oddsratios in Example mentioned below).

A kit for detecting the genotype, comprising two or more sets of nucleicacids selected from the group consisting of the following (11) to (15):

-   -   (11) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing a part of polymorphism        at position −1171 of the stromelysin 1 gene and which consists        of a sense primer that specifically hybridizes the partial DNA        region containing the corresponding sequence of the stromelysin        1 gene in which five A successively exist in the 3′ direction        from the position −1171, and/or a sense primer that specifically        hybridizes the partial DNA region containing the corresponding        sequence of the streomelysin 1 in which six A successively exist        in the 3′ direction from the position −1171, and an antisense        primer that specifically hybridizes a part of region of the        stromelysin 1 gene;    -   (12) a set of nucleic acids consisting of a pair of primers        which are designed to specifically amplify the partial DNA        region containing a part of polymorphism at position −668 of the        plasminogen activator inhibitor 1 gene, as well as a probe that        specifically hybridizes the partial DNA region containing the        corresponding sequence in the plasminogen activator inhibitor 1        gene in which four G successively exist in the 3′ direction from        the position −668, and/or a probe that specifically hybridizes        the partial DNA region containing the corresponding sequence in        the plasminogen activator inhibitor 1 gene in which five G        successively exist in the 3′ direction from the position −668;    -   (13) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        1018 of the glycoprotein Ibα gene and which consists of a sense        primer that specifically hybridizes the partial DNA region        containing the base at position 1018 of the glycoprotein Ibα        gene in which the base at position 1018 is C, and/or a sense        primer that specifically hybridizes the partial DNA region of        the glycoprotein Ibα gene in which the base at position 1018 is        T, and an antisense primer that specifically hybridizes a part        of region of the glycoprotein Ibα gene;    -   (14) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        584 of the paraoxonase gene and which consists of a sense primer        that specifically hybridizes the partial DNA region containing        the base at position 584 of the paraoxonase gene in which the        base at position 584 is G, and/or a sense primer that        specifically hybridizes the partial DNA region of the        paraoxonase gene in which the base at position 584 is A, and an        antisense primer that specifically hybridizes a part of region        of the paraoxonase gene; and    -   (15) a set of nucleic acids which is designed to specifically        amplify the partial DNA region containing the base at position        4070 of the apolipoprotein E gene and which consists of a sense        primer that specifically hybridizes the partial DNA region        containing the base at position 4070 of the apolipoprotein E        gene in which the base at position 4070 is C, and/or a sense        primer that specifically hybridizes the partial DNA region of        the apolipoprotein E gene in which the base at position 4070 is        T, and an antisense primer that specifically hybridizes a part        of region of the apolipoprotein E gene;

In the above mention, kits are constructed by selecting two or more setsof nucleic acids from the group consisting of (11) to (15). However,kits may be constructed by making a group consisting of two or more setsof nucleic acids arbitrarily selected from (11) to (15) and selectingtwo or more sets of nucleic acids from such a group. For example, kitsare constructed by selecting two or more nucleic acids from the groupconsisting of (11), (12), (14) and (15) (sets of nucleic acids forpolymorphism analysis with odds ratio of one or more in Examplementioned below), or kits are constructed by selecting two or more setsof nucleic acids from the group consisting of (11), (12) and (15) (setsof nucleic acids for polymorphism analysis with three highest oddsratios in Example mentioned below).

A kit for detecting the genotype, comprising the following a set ofnucleic acids,

a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 4070 of theapolipoprotein E gene and which consists of a sense primer thatspecifically hybridizes the partial DNA region containing the base atposition 4070 of the apolipoprotein E gene in which the base at position4070 is C, and/or a sense primer that specifically hybridizes thepartial DNA region of the apolipoprotein E gene in which the base atposition 4070 is T, and an antisense primer that specifically hybridizesa part of region of the apolipoprotein E gene.

A kit for detecting the genotype comprising two or more sets of nucleicacids selected from the group consisting of the following (1) to (10);

-   -   (1) a set of nucleic acids which consists of a first nucleic        acid that specifically hybridizes a partial region containing        the base at position 1019 in the antisense strand of the        connexin 37 gene whose base at position 1019 is C and that is        labeled with a first labeling substance, of a second nucleic        acid that specifically hybridizes a partial region containing        the base at position 1019 in the antisense strand of the        connexin 37 gene whose base at position 1019 is T and that is        labeled with a second labeling substance, and of a third nucleic        acid that specifically hybridizes a partial region of the sense        strand of the connexin 37 gene and that can specifically amplify        the partial DNA region containing the base at position 1019 of        the connexin 37 in concurrent use with the above first or second        nucleic acid;    -   (2) a set of nucleic acids which consists of a first nucleic        acid that specifically hybridizes a partial region containing        the base at position −863 in the sense strand of the tumor        necrosis factor α gene whose base at position −863 is C and that        is labeled with a first labeling substance, of a second nucleic        acid that specifically hybridizes a partial region containing        the base at position −863 in the sense strand of the tumor        necrosis factor α gene whose base at position −863 is A and that        is labeled with a second labeling substance, and of a third        nucleic acid that specifically hybridizes a partial region of        the antisense strand of the tumor necrosis factor α gene and        that can specifically amplify the partial DNA region containing        the base at position −863 of the tumor necrosis factor α in        concurrent use with the above first or second nucleic acid;    -   (3) a set of nucleic acids which consists of a first nucleic        acid that specifically hybridizes a partial region containing        the base at position 242 in the sense strand of the NADH/NADPH        oxidase p22 phox gene whose base at position 242 is C and that        is labeled with a first labeling substance, of a second nucleic        acid that specifically hybridizes a partial region containing        the base at position 242 in the sense strand of the NADH/NADPH        oxidase p22 phox gene whose base at position 242 is T and that        is labeled with a second labeling substance, and of a third        nucleic acid that specifically hybridizes a partial region of        the antisense strand of the NADH/NADPH oxidase p22 phox gene and        that can specifically amplify the partial DNA region containing        the base at position 242 of the NADH/NADPH oxidase p22 phox in        concurrent use with the above first or second nucleic acid;    -   (4) a set of nucleic acids which consists of a first nucleic        acid that specifically hybridizes a partial region containing        the base at position −6 in the sense strand of the        angiotensinogen gene whose base at position −6 is G and that is        labeled with a first labeling substance, of a second nucleic        acid that specifically hybridizes a partial region containing        the base at position −6 in the sense strand of the        angiotensinogen gene whose base at position −6 is A and that is        labeled with a second labeling substance, and of a third nucleic        acid that specifically hybridizes a partial region of the        antisense strand of the angiotensinogen gene and that can        specifically amplify the partial DNA region containing the base        at position −6 of the angiotensinogen in concurrent use with the        above first or second nucleic acid;    -   (5) a set of nucleic acids which consists of a first nucleic        acid that specifically hybridizes a partial region containing        the base at position −219 in the antisense strand of the        apolipoprotein E gene whose base at position −219 is G and that        is labeled with a first labeling substance, of a second nucleic        acid that specifically hybridizes a partial region containing        the base at position −219 in the antisense strand of the        apolipoprotein E gene whose base at position −219 is T and that        is labeled with a second labeling substance, and of a third        nucleic acid that specifically hybridizes a partial region of        the sense strand of the apolipoprotein E gene and that can        specifically amplify the partial DNA region containing the base        at position −219 of the apolipoprotein E in concurrent use with        the above first or second nucleic acid;    -   (6) a set of nucleic acids which consists of a first nucleic        acid that specifically hybridizes a partial region containing        the base at position 994 in the antisense strand of the        platelet-activating factor acetylhydrolase gene whose base at        position 994 is G and that is labeled with a first labeling        substance, of a second nucleic acid that specifically hybridizes        a partial region containing the base at position 994 in the        antisense strand of the platelet-activating factor        acetylhydrolase gene whose base at position 994 is T and that is        labeled with a second labeling substance, and of a third nucleic        acid that specifically hybridizes a partial region of the sense        strand of the platelet-activating factor acetylhydrolase gene        and that can specifically amplify the partial DNA region        containing the base at position 994 of the platelet-activating        factor acetylhydrolase gene in concurrent use with the above        first or second nucleic acid;    -   (7) a set of nucleic acids which consists of a first nucleic        acid that specifically hybridizes a partial region containing        the base at position −482 in the antisense strand of the        apolipoprotein C-III gene whose base at position −482 is C, of a        second nucleic acid that specifically hybridizes a partial        region containing the base at position −482 in the antisense        strand of the apolipoprotein C-III gene whose base at position        −482 is T, of a third nucleic acid that specifically hybridizes        a partial region of the sense strand of the apolipoprotein C-III        gene and that can specifically amplify the partial DNA region        containing the base at position −482 of the apolipoprotein C-III        gene in concurrent use with the above first or second nucleic        acid, of the fourth nucleic acid that specifically hybridizes        the nucleic acid amplified by the use of the first and third        nucleic acids; and of the fifth nucleic acid that specifically        hybridizes the nucleic acid amplified by the use of the second        and third nucleic acids;    -   (8) a set of nucleic acids which consists of a first nucleic        acid that specifically hybridizes a partial region containing        the base at position 1186 in the antisense strand of the        thrombospondin 4 gene whose base at position 1186 is G and that        is labeled with a first labeling substance, of a second nucleic        acid that specifically hybridizes a partial region containing        the base at position 1186 in the antisense strand of the        thrombospondin 4 gene whose base at position 1186 is C and that        is labeled with a second labeling substance, and of a third        nucleic acid that specifically hybridizes a partial region of        the sense strand of the thrombospondin 4 gene and that can        specifically amplify the partial DNA region containing the base        at position 1186 of the thrombospondin 4 gene in concurrent use        with the above first or second nucleic acid;    -   (9) a set of nucleic acids which consists of a first nucleic        acid that specifically hybridizes a partial region containing        the base at position −819 in the antisense strand of the        interleukin-10 gene whose base at position −819 is T, of a        second nucleic acid that specifically hybridizes a partial        region containing the base at position −819 in the antisense        strand of the interleukin-10 gene whose base at position −819 is        C, of a third nucleic acid that specifically hybridizes a        partial region of the sense strand of the interleukin-10 gene        and that can specifically amplify the partial DNA region        containing the base at position −819 of the interleukin-10 gene        in concurrent use with the above first or second nucleic acid,        of the fourth nucleic acid that specifically hybridizes the        nucleic acid amplified by the use of the first and third nucleic        acids; and of the fifth nucleic acid that specifically        hybridizes the nucleic acid amplified by the use of the second        and third nucleic acids; and    -   (10) a set of nucleic acids which consists of a first nucleic        acid that specifically hybridizes a partial region containing        the base at position −592 in the sense strand of the        interleukin-10 gene whose base at position −592 is A and that is        labeled with a first labeling substance, of a second nucleic        acid that specifically hybridizes a partial region containing        the base at position −592 in the sense strand of the        interleukin-10 gene whose base at position −592 is C and that is        labeled with a second labeling substance, and of a third nucleic        acid that specifically hybridizes a partial region of the        antisense strand of the interleukin-10 gene and that can        specifically amplify the partial DNA region containing the base        at position −592 of the interleukin-10 gene in concurrent use        with the above first or second nucleic acid.

In the above mention, kits are constructed by selecting two or more setsof nucleic acids from the group consisting of (1) to (10). However, kitsmay be constructed by making a group consisting of two or more sets ofnucleic acids arbitrarily selected from (1) to (10) and selecting two ormore sets of nucleic acids from such a group. For example, kits may beconstructed by selecting two or more sets of nucleic acids from thegroup consisting of (1), (5), (6), (8), (9) and (10) (sets of nucleicacids for polymorphism analysis with odds ratio of one or more inExample mentioned below), or kits may be constructed by selecting two ormore sets of nucleic acids from the group consisting of (1), (5), (6),(8) and (9) (sets of nucleic acids for polymorphism analysis with fivehighest odds ratios in Example mentioned below).

A kit for detecting the genotype, comprising two or more sets of nucleicacids selected from the group consisting of the following (11) to (15):

-   -   (11) a set of nucleic acids which consists of a first nucleic        acid that specifically hybridizes a partial region containing a        sequence which corresponds to the part of sequence in the        antisense strand of the stromelysin 1 gene in which five A        successively exist in the 3′ direction from the position −1171        and that is labeled with a first labeling substance, of a second        nucleic acid that specifically hybridizes a partial region        containing a sequence which corresponds to the part of sequence        in the antisense strand of the stromelysin 1 gene in which six A        successively exist in the 3′ direction from the position −1171        and that is labeled with a second labeling substance, and of a        third nucleic acid that specifically hybridizes a partial region        of the sense strand of the stromelysin 1 gene and that can        specifically amplify the partial DNA region containing the base        at position −1171 of the stromelysin 1 gene in concurrent use        with the above first or second nucleic acid;    -   (12) a set of nucleic acids which consists of a pair of nucleic        acids (a first nucleic acid and a second nucleic acid) that is        designed to specifically amplify the partial DNA region        containing a part of polymorphism at position -668 of the        plasminogen activator inhibitor 1 gene, of a third nucleic acid        that specifically hybridizes the nucleic acid which is obtained        by amplification using plasminogen activator inhibitor 1 gene in        which four G successively exist in the 3′ direction from the        position −668 as a template and the set of nucleic acids, and of        a four nucleic acid that specifically hybridizes a nucleic acid        which is obtained by amplification using plasminogen activator        inhibitor 1 gene in which five G successively exist in the 3′        direction from the position −668 as a template and the set of        nucleic acids;    -   (13) a set of nucleic acids which consists of a first nucleic        acid that specifically hybridizes a partial region containing a        base which corresponds to the base at position 1018 in the        antisense strand of the glycoprotein Ibα gene whose base at        position 1018 is C and that is labeled with a first labeling        substance, of a second nucleic acid that specifically hybridizes        a partial region containing a base which corresponds to the base        at position 1018 in the antisense strand of the glycoprotein Ibα        gene whose base at position 1018 is T and that is labeled with a        second labeling substance, and of a third nucleic acid that        specifically hybridizes a partial region of the sense strand of        the glycoprotein Ibα gene and that can specifically amplify the        partial DNA region containing the base at position 1018 of the        glycoprotein Ibα gene in concurrent use with the above first or        second nucleic acid;    -   (14) a set of nucleic acids which consists of a first nucleic        acid that specifically hybridizes a partial region containing a        base which corresponds to the base at position 584 in the        antisense strand of the paraoxonase gene whose base at position        584 is G and that is labeled with a first labeling substance, of        a second nucleic acid that specifically hybridizes a partial        region containing a base which corresponds to the base at        position 584 in the antisense strand of the paraoxonase gene        whose base at position 584 is A and that is labeled with a        second labeling substance, and of a third nucleic acid that        specifically hybridizes a partial region of the sense strand of        the paraoxonase gene and that can specifically amplify the        partial DNA region containing the base at position 584 of the        paraoxonase gene in concurrent use with the above first or        second nucleic acid; and    -   (15) a set of nucleic acids which consists of a first nucleic        acid that specifically hybridizes a partial region containing a        base which corresponds to the part of sequence in the antisense        strand of the apolipoprotein E gene whose base at position 4070        is C and that is labeled with a first labeling substance, of a        second nucleic acid that specifically hybridizes a partial        region containing a base which corresponds to the part of        sequence in the antisense strand of the apolipoprotein E gene        whose base at position 4070 is T and that is labeled with a        second labeling substance, and of a third nucleic acid that        specifically hybridizes a partial region of the sense strand of        the glycoprotein Ibα gene and that can specifically amplify the        partial DNA region containing the base at position 4070 of the        apolipoprotein E gene in concurrent use with the above first or        second nucleic acid.

In the above mention, kits are constructed by selecting two or more setsof nucleic acids from the group consisting of (11) to (15). However,kits may be constructed by making a group consisting of two or more setsof nucleic acids arbitrarily selected from (11) to (15) and selectingtwo or more sets of nucleic acids from such a group. For example, kitsare constructed by selecting two or more nucleic acids from the groupconsisting of (11), (12), (14) and (15) (sets of nucleic acids forpolymorphism analysis with odds ratio of one or more in Examplementioned below), or kits are constructed by selecting two or morenucleic acids from the group consisting of (11), (12) and (15) (sets ofnucleic acids for polymorphism analysis with three highest odds ratiosin Example mentioned below).

A kit for detecting the genotype, comprising the following sets ofnucleic acids:

a set of nucleic acids which consists of a first nucleic acid thatspecifically hybridizes a partial region containing a base whichcorresponds to the part of sequence in the antisense strand of theapolipoprotein E gene whose base at position 4070 is C and that islabeled with a first labeling substance, of a second nucleic acid thatspecifically hybridizes a partial region containing a base whichcorresponds to the part of sequence in the antisense strand of theapolipoprotein E gene whose base at position 4070 is T and that islabeled with a second labeling substance, and of a third nucleic acidthat specifically hybridizes a partial region of the sense strand of theglycoprotein Ibα gene and that can specifically amplify the partial DNAregion containing the base at position 4070 of the apolipoprotein E genein concurrent use with the above first or second nucleic acid.

In the above-mentioned kits, one or two or more of reagents (buffer,reagent for reaction, and reagent for detection, etc.) may be combinedin response to the usage of the kit.

The present invention is hereinafter explained in more detail by way ofExamples.

EXAMPLE 1 Selection of Gene Polymorphism

By using several kinds of common databases including PubMed [NationalCenter for Biological Information (NCBI)], Online Mendelian inheritancein Men (NCBI), Single Nucleotide Polymorphism (NCBI), etc., from acomprehensive viewpoint including vascular biology, platelet-leucocytebiology, congealing fibrinogenolysis system, a metabolic factor such aslipid, sugar, etc., 71 genes which were estimated to be associated withcoronary arteriosclerosis, coronary artery spasm, hypertension,diabetes, hyperlipidemia, etc. were extracted from genes which had beenpreviously reported. Furthermore, in the polymorphisms existing in thesegenes, 112 polymorphisms including polymorphisms which exist in promoterregions or exons, or polymorphisms which were located in splice donorsites or acceptor sites and which were expected to be associated withthe functional changes of gene products were selected (FIGS. 1 and 2).

EXAMPLE 2 Determination of Gene Polymorphism

Subjects were 5061 Japanese males and females (3309 males and 1752females) who visited as outpatients or were hospitalized in 15participating institutes between July 1994 and December 2001. 2819subjects (2003 males and 816 females) had myocardial infarction. Allsubjects were subjected to coronary angiography and leftventriculography. Diagnosis of myocardial infarction was carried outbased on electrocardiographic change and increases in serum CK, GOT andLDH. Confirmed diagnosis of myocardial infarction was determined basedon abnormality in wall motion in left ventriculography and stenosis ofleft main coronary artery or major coronary arteries correspondingthereto.

Controls were 2242 people (1306 males and 936 females) who visited theparticipating institutes and had at least one of the conventional riskfactors of coronary artery diseases, i.e., smoking (10 cigarettes ormore per day), obesity (body mass index ≧26 kg/m²), hypertension(systolic blood pressure ≧140 mmHg or/and diastolic blood pressure ≧90mmHg), diabetes (fasting blood sugar ≧126 mg/dL or/and hemoglobin Alc≧6.5%), hyperlipidemia (total cholesterol in serum ≧220 mg/dL),hyperuricemia (male: uric acid ≧7.7 mg/dL, female: uric acid ≧5.5 mg/dL)but did not have coronary artery disease. In these controls, restingelectrocardiogram showed normal, and also in exercise tolerance test, nochange showing myocardial ischemia was observed.

From each of the subjects, 7 mL of venous blood was collected in a tubecontaining 50 mmol/L EDTA-2Na and genome DNA was extracted by using DNAextraction kit (Qiagen, Chatsworth, Calif.). 71 candidate genes 112polymorphisms were determined by allele specific primer-probemeasurement system (Toyobo Gene Analysis, Tsuruga, Japan) byfluorescence method and spectrometry (see FIGS. 3 and 4). DNAfragment-containing a polymorphism site was amplified by polymerasechain reaction (PCR) by using two kinds of allele specific sense primers(or antisense primers) whose 5′ end were labeled with fluoresceinisothiocyanate (FITC) or Texas red (TxR) and an antisense primer (or asense primer) whose 5′ end was labeled with biotin. Alternatively, DNAfragment containing polymorphism site was amplified by PCR by using twokinds of allele specific sense primer and antisense primer whose 5′ endwas labeled with biotin or by using a sense primer and antisense primerwhose 5′ end was labeled with biotin. The reaction solution (25 μL)contained 20 ng of DNA, 5 pmol of each primer, 0.2 mmol/L of eachdeoxynucleoside triphosphate (DATP, dGTP, dCTP and dTTP), 1-4 mmol/L ofMgCl₂, 1 U DNA polymerase (rTaq or KODplus; Toyobo Co., Ltd. Osaka,Japan), and each DNA polymerase buffer was used. Amplification protocolincluded: initial degeneration at 95° C. for 5 minutes; 35-45 cycles ofdegeneration at 95° C. for 30 minutes, annealing at 55-67.5° C. for 30seconds, and extension at 72° C. for 30 seconds; and final extension at72° C. for 2 minutes.

In the determination of the genotype by fluorescent method, amplifiedDNA was incubated in a solution containing streptavidin binding magneticbeads in a 96-well plate at room temperature. This plate was disposed ona magnetic stand and supernatant was collected from each well andtransferred into each well of the 96-well plate containing 0.01 M NaOH,followed by measuring fluorescence by microplate reader at excitationwavelength and emission wavelength of 485 nm and 538 nm for FITC and atexcitation wavelength and emission wavelength of 584 nm and 612 nm forTxR. Furthermore, in the determination of the genotype by spectrometry,amplified DNA was denatured by 0.3 M NaOH and hybridized by using ahybridization buffer containing any of allele specific probe fixed tothe bottom surface of each well of the 96-well plate and 35-40%formamide at 37° C. for 30 minutes. The well was thoroughly washed andthen alkaline phosphatase binding streptavidin was added to each welland the plate was shaken at 37° for 15 minutes. The well was washedagain and a solution containing 0.8 mM2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium(monosodium salt) and 0.4 mM 5-bromo-4-chloro-3-indolyl phosphatep-toluidine salt was added, followed by measuring the absorbance (450nm).

In order to confirm the accuracy of the determination of the genotype,DNA samples of 50 people were selected at random, and the samples weresubjected to PCR-restriction fragment length polymorphism (PCR-RFLP)method or direct sequence determination for nucleic acid of PCR product.In any samples, the genotype determined by the allele specificprimer-probe measurement system were the same as those determined byPCR-polymerase chain reaction-restriction fragment length polymorphismmethod or direct determination method of DNA sequence.

Note here that statistical analysis in the following association studywas carried out as follows. First of all, data were shown inaverage±standard deviation. Comparison of clinical data was carried outbetween patients with myocardial infarction and controls by using anunpaired Student's t test or a Mann-Whitney U test. Data in three groupswere compared by way of a one-way analysis of variance and a Scheffe'spost-hoc test or a Kruskal-Wallis test. Qualitative data were tested bya chi-square test. The allele frequency was estimated by a gene countingmethod and its deviation from the Hardy-Weinberg equilibrium was testedby a chi-square test. Furthermore, a multivariate logistic regressionanalysis in which risk factors were corrected was carried out.Myocardial infarction was used as a dependent variable, and age, bodymass index (BMI), smoking condition (0=no smoking, 1=smoking), metabolicfactor (0=without histories of hypertension, diabetes,hypercholesteremia, hyperuricemia, 1 =with histories above) and genotypeof respective polymorphisms were used as independent variables. Therespective genotypes were analyzed for dominant, recessive, additivegenetic models, and P value, odds ratio, 95% confidence interval (CI)were calculated. In the combination of analysis of the genotype, oddsratio of each the genotype was calculated by a stepwise forwardselection method of logistic regression analysis.

EXAMPLE 3 Selection of Polymorphism Associated with MyocardialInfarction and Development of Method for Diagnosing MyocardialInfarction

Firstly, screening related analyses regarding 71 genes 112 polymorphismswere carried out for 451 males (myocardial infarction: 219, control:232) and 458 females (myocardial infarction: 226, control: 232). Thesecases were selected at random from entire 5061 cases.

Background data of 909 people (451 males and 458 females) subjected toscreening related analyses by the above-mentioned method were shown inFIG. 5. In males, no significant difference was found in age, BMI, andfrequency of conventional risk factors of coronary artery disease suchas smoking, hypertension, diabetes, hypercholesterolemia, hyperuricemia,etc. between myocardial infraction group and control group. In females,no significant difference was found in age, BMI, and frequency ofhypercholesterolemia, hyperuricemia, etc. was not found betweenmyocardial infraction group and control group, but the prevalence ofsmoking or diabetes was significantly higher in myocardial infarctiongroup as compared with the control group. In the screening-relatedanalysis between 112 polymorphisms and myocardial infarction, by themultivariate logistic regression analysis in which age, BMI, andconventional risk factors for coronary artery disease such as smoking,hypertension, diabetes, hypercholesterolemia, hyperuricemia werecorrected, 19 single nucleotide polymorphisms (SNP) in males and 18single nucleotide polymorphisms (SNP) in females were shown to beassociated with myocardial infarction (FIG. 6). Note here that in thescreening-related analyses, category, in which P value <0.1 in logisticregression analysis shows to have relation, was employed. In these SNPs,four SNPs were related to myocardial infarction in both males andfemales and other SNPs were related to myocardial infarction in eitherone of males or females.

Then, the determination of the genotype of these polymorphisms wascarried out in the rest 4152 cases (male myocardial infarction subjects:1784, male control: 1074, female myocardial infarction subjects: 590 andfemale control: 704). A large scale association study of thesepolymorphisms and myocardial infarction was carried out in total 5061cases (male myocardial infarction subjects: 2003, male control: 1306,female myocardial infarction subjects: 816 and female control: 936).

FIG. 7 shows background data of the total 5061 cases (3309 males and1752 females) in the large scale association study. In males, nosignificant difference was found in age, BMI and frequency of smokingbetween the myocardial infarction group and the control group, but theprevalence of hypertension or hyperuricemia was significantly lower inmyocardial infarction group as compared with the control group, and theprevalence of diabetes or hypercholesterolemia was significantly higherin myocardial infarction group as compared with the control group. Infemales, no significant difference in age or the prevalence ofhypertension was found between the infarction group and the controlgroup, but BMI and the prevalence of smoking, diabetes,hypercholesterolemia,or hyperuricemia was significantly higher inmyocardial infarction group as compared with the control group. In alarge scale association study of myocardial infarction in the males (19SNP) and females (18 SNP), by the multivariable logistic regressionanalysis in which age, BMI, and frequency of smoking, hypertension,diabetes, hypercholesterolemia, hyperuricemia were corrected, 10 SNPs inmales and 5 SNPs in females showed significant relation to themyocardial infarction (P<0.05 in either of dominant or recessive genemodel) (FIG. 8). Distribution of the genotypes and results of logisticregression analysis are shown in FIGS. 8 and 9, respectively.

In this Example, a stepwise forward selection method of multivariatelogistic regression analysis was carried out (see FIG. 10). This methodemployed dominant or recessive genetic model based on P value inrelation to the myocardial infarction of the respective SNPs shown inFIG. 9. FIG. 10 shows gene locus on the chromosome of these genes. The−819T→C polymorphism and −592A→C polymorphism in the interleukin-10 werein linkage disequilibrium [pairwise linkage disequilibrium coefficient,D′ (D/D_(max)), of 0.406; standardized linkage disequilibriumcoefficient, .r, of 0.396; P<0.0001, chi-square test]. Gene loci oftumor necrosis factor α gene and platelet-activating factoracetylhydrolase gene were in proximity to each other, but norelationship was found in distribution of the genotype of polymorphismsof the both genes. Similarly, gene loci of plasminogen activatorinhibitor 1 gene and paraoxonase were in proximity to each other, but norelationship was found in distribution of the genotype of polymorphismsof the both genes.

The odds ratio of contraction with myocardial infarction by thecombination of the genotype calculated by the stepwise forward selectionmethod was shown in FIG. 11 and FIG. 13(A) as to males, and in FIG. 12and FIG. 13(B) as to females. In males, maximum odds ratio was 4.50 inthe genotype combination of five SNPs (TSP4 (1186G→C) polymorphism,connexin 37 (1019C→T) polymorphism, PAF acetylhydrolase (994G→T)polymorphism, angiotensinogen (−6G→A) polymorphism, tumor necrosisfactor α (−863C→A) polymorphism) (see FIG. 11 and FIG. 13(A)). In thecase where further five SNPs (SNP (NADH/NADPH oxidase p22 phox (242C→T)polymorphism, Apo E (−219G→T) polymorphism, Apo C-III (−482C→T)polymorphism, IL-10 (−819T→C) polymorphism, IL-10 (−592A→C)polymorphism) were added and SNPs are 10 in total, maximum odds ratiowas 11.26 (see FIG. 10 and FIG. 13(A)). In females, by the combinationof five SNP (Apo E (4070C→T) polymorphism, glycoprotein Ibα (1018C→T)polymorphism, stromelysin 1 (−1171/5A.6A) polymorphism, PAIL(−668/4G-5G) polymorphism, paraoxonase (584G→A) polymorphism)), maximumodds ratio was 88.51 (see FIG. 12 and FIG. 13(B)).

As mentioned above, the present inventors have investigated therelationship between myocardial infarction and 112 polymorphisms whichwere selected from 71 candidate genes; and identified 10 SNPs (in males)and five SNPs (in females) which were associated with myocardialinfarction by way of a large scale association study of 5061 cases.Furthermore, by the stepwise forward selection method of multivariatelogistic regression analysis, a method for diagnosing the risk ofmyocardial infarction (genetic risk diagnostic system) presenting themaximum odds ratio of 11.26 in males and maximum odds ratio of 88.51 wasdeveloped.

Main causes of myocardial infarction is arteriosclerotic coronary arterydisease, which may cause hemodynamically significant stenosis in theinternal diameter of artery to cause abnormalities in regulation ofvasoconstriction and vasodilator action. As a result, rupture ofarterial sclerosis lesion or thrombogenesis is likely to occur. Thepresent inventors have selected 71 candidate genes based on thecomprehensive viewpoint including vascular biology, platelet-leukocytebiology, coagulation and fibrinolysis system, a metabolic factor such aslipid, sugar, etc. In fact, a group of genes related to myocardialinfarction had various roles in development condition. That is to say,vascular biology (connexin 37, NADH/NADPH oxidase p22 phox, andthrombospondin 4), vascular inflammation (tumor necrosis factor-α,platelet-activating factor acetylhydrolase, and interleukin-10),hypertension (angiotensinogen), lipid metabolism (apolipoprotein E andC-III and paraoxonase), function of platelet (glycoprotein Iba), matrixmetabolism (stromelysin-1), fibrinolytic system (PAI-1), and the like,are included (Boerma M, Forsberg L, van Zeijl L, et al. A geneticpolymorphism in connexin 37 as a prognostic marker for atheroscleroticplaque development. J Intern Med 1999;246: 211-218, Inoue N, KawashimaS, Kanazawa K, Yamada S, Akita H, Yokoyama M. Polymorphism of theNADH/NADPH oxidase p22 phox gene in patients with coronary arterydisease. Circulation 1998;97: 135-137, Topol E J, McCarthy J, Gabriel S,et al. Single nucleotide polymorphisms in multiple novel thrombospondingenes may be associated with familial premature myocardial infarction.Circulation 2001;104: 2641-2644, Skoog T, van't Hooft F M, Kallin B, etal. A common functional polymorphism (C→A substitution at position −863)in the promoter region of the tumor necrosis factor-α (TNF-α) geneassociated with reduced circulating level of TNF-α. Hum Mol Genet1999;8: 1443-1449, Yamada Y, Ichihara S, Fujimura T, Yokota M.Identification of the G⁹⁹⁴→T missense mutation in exon 9 of the plasmaplatelet-activating factor acetylhydrolase gene as an independent riskfactor for coronary artery disease in Japanese men. Metabolism 1998;47:177-181, Koch W, Kastrati A, Bottiger C, Mehilli J, von Beckerath N,Schomig A. Interleukin-10 and tumor necrosis factor gene polymorphismsand risk of coronary artery disease and myocardial infarction.Atherosclerosis 2001;159: 137-144, Inoue I, Nakajima T, Williams C S, etal. A nucleotide substitution in the promoter of human angiotensinogenis associated with essential hypertension and affects basaltranscription in vitro. J Clin Invest 1997;99: 1786-1797, Lambert J-C,Brousseau T, Defosse V, et al. Independent association of an APOE genepromoter polymorphism with increased risk of myocardial infarction anddecreased APOE plasma concentrations-the ECTIM study. Hum Mol Genet2000;9: 57-61, Eto M, Watanabe K, Makino I. Increased frequency ofapolipoprotein epsilon 2 and epsilon 4 alleles in patients with ischemicheart disease. Clin Genet 1989;36: 183-188., Ruiz J, Blanche H, James RW, et al. Gln-Arg192 polymorphism of paraoxonase and coronary heartdisease in type 2 diabetes. Lancet 1995;346: 869-72, Murata M, MatsubaraY, Kawano K, et al. Coronary artery disease and polymorphisms in areceptor mediating shear stress-dependent platelet activation.Circulation 1997;96: 3281-3286, Eriksson P, Kallin B, van't Hooft F M,Bavenholm P, Hamsten A. Allele-specific increase in basal transcriptionof the plasminogen-activator inhibitor 1 gene is associated withmyocardial infarction. Proc Natl Acad Sci USA 1995;92: 1851-1855, Ye S,Watts G F, Mandalia S, Humphries S E, Henney A M. Preliminary report:genetic variation in the human stromelysin promoter is associated withprogression of coronary atyherosclerosis. Br Heart J 1995; 73: 209-215).The present inventors have investigated 112 gene polymorphisms in 909cases and investigated 19 SNPs in 2858 male cases and 18 SNPs in 1294female cases. As a result, the present inventors determined 179,402genotypes in total. The number of determined genes is the largest amongthose reported previously in the last relation analysis of genepolymorphisms. The method for diagnosing the risk of myocardialinfarction shown in the above-mentioned Examples had maximum odds ratioof 11.26 in males and 88.51 in females, which are also maximum amongthose in the previously reported relation analysis.

In 15 SNPs associated with myocardial infarction, 4070T-C (Arg158Cys)polymorphism of the apolipoprotein E gene exhibited a maximum odds ratioas female myocardial infarction. Apolipoprotein E is a main component ofchylomicron and very low density lipoprotein (VLDL) remnant andfunctions as ligand when these lipoporoteins are taken by receptors inthe liver (Mahley R W. Apolipoprotein E: cholesterol transport proteinwith expanding role in cell biology. Science 1998;240: 622-630). 158Cys(e2) allele of apolipoprotein E gene causes abnormal binding to thereceptor in the liver (Schneider W J, Kovanen P T, Brown M S, et al.Familial dysbetalipoproteinemia. Abnormal binding of mutantapolipoprotein E to low density lipoprotein receptors of humanfibroblasts and membranes from liver and adrenal of rats, rabbits, andcows. J Clin Invest 1981;68: 1075-1085), and the removal from plasma isdelayed (Gregg R E, Zech L A, Schaefer E J, Brewer H B Jr. Type IIIhyperlipoproteinemia: defective metabolism of an abnormal apolipoproteinE. Science 1981;211: 584-586). Most of familial dysbetalipoproteinemia(FD, or III type hyperlipoproteinemia) patients have homozygote ofArg158Cys polymorphism (Breslow J L, Zannis V I, SanGiacomo T R, Third JL, Tracy T, Glueck C J. Studies of familial type IIIhyperlipoproteinemia using as a genetic marker the apo E phenotype E2/2.J Lipid Res 1982;23: 1224-1235). However, since only 1% to 4% of158Cys/Cys homozygote develops familial dysbetalipoproteinemia, it isthought that other genetic factors or environmental factors arenecessary to this disease. Accumulation of arteriosclerosis remnantlipoprotein (β-VLDL) in plasma in familial dysbetalipoproteinemiapatients (Mahley R W. Apolipoprotein E: cholesterol transport proteinwith expanding role in cell biology. Science 1998;240: 622-630) or amouse over-expressing human 158Cys/Cys (Sullivan P M, Mezdour H,Quarfordt S H, Maeda N. Type III hyperlipoproteinemia and spontaneousatherosclerosis in mice resulting from gene replacement of mouse Apoewith human APOE*2. J Clin Invest 1998;102: 130-135) is found to promotethe development of arteriosclerosis. Eto et al. have reported that e2(158Cys) allele is associated with coronary artery disease in Japanesemales (odds ratio=2.44, e2 allele pair, e3/e3 type) and females (oddsratio=3.03) (Eto M, Watanabe K, Makino I. Increased frequency ofapolipoprotein epsilon 2 and epsilon 4 alleles in patients with ischemicheart disease. Clin Genet 1989;36: 183-188). The conclusion of thepresent inventors that TT type (158Cys/Cys) is a risk factor formyocardial infarction agrees with the conclusion of Eto et al.

Some of SNPs investigated in the above-mentioned Examples may be in alinkage disequilibrium with SNPs of genes actually associated with thedevelopment of myocardial infarction existing in the vicinity thereof.However, nine genes in male and five genes in females are shown to besusceptible gene loci of myocardial infarction. Furthermore, combinationof genotypes enables high reliable and predictability diagnostic method.Thus, diagnostic method of the present invention can be expected tocontribute to primary prevention of myocardial infarction and theimprovement in quality of life of middle and old aged persons as well asthe reduction of health care cost.

The present invention is not limited to the description of the aboveembodiments. A variety of modifications, which are within the scopes ofthe following claims and which are achieved easily by a person skilledin the art, are included in the present invention.

Hereinafter, the following matters are disclosed.

-   -   11. A method for detecting the genotype, comprising the        following step (a1),        -   (a1) analyzing the following polymorphisms (1) to (10) in a            nucleic acid sample:            -   (1) polymorphism at the base number position 1019 of the                connexin 37 gene;            -   (2) polymorphism at the base number position −863 of the                tumor necrosis factor α gene;            -   (3) polymorphism at the base number position 242 of the                NADH/NADPH oxidase p22 phox gene;            -   (4) polymorphism at the base number position −6 of the                angiotensinogen gene;            -   (5) polymorphism at the base number position −219 of the                apolipoprotein E gene;            -   (6) polymorphism at the base number position 994 of the                platelet-activating factor acetylhydrolase gene;            -   (7) polymorphism at the base number position −482 of the                apolipoprotein C-III gene;            -   (8) polymorphism at the base number position 1186 of the                thrombospondin 4 gene;            -   (9) polymorphism at the base number position −819 of the                interleukin-10 gene; and            -   (10) polymorphism at the base number position −592 of                the interleukin-10 gene.    -   12. A method for detecting the genotype, comprising the        following step (b1),        -   (b1) analyzing the following polymorphisms (11) to (15) in a            nucleic acid sample:            -   (11) polymorphism at the base number position −1171 of                the stromelysin 1 gene;            -   (12) polymorphism at the base number position −668 of                the plasminogen activator inhibitor-1 gene;            -   (13) polymorphism at the base number position 1018 of                the glycoprotein Ibα gene;            -   (14) polymorphism at the base number position 584 of the                paraoxonase gene: and            -   (15) polymorphism at the base number position 4070 of                the apolipoprotein E gene.    -   13. A method for diagnosing the risk of myocardial infarction,        comprising the following (i) to (iii):        -   (i) a step of analyzing the following polymorphisms (1)            to (10) in a nucleic acid sample:            -   (1) polymorphism at the base number position 1019 of the                connexin 37 gene;            -   (2) polymorphism at the base number position −863 of the                tumor necrosis factor α gene;            -   (3) polymorphism at the base number position 242 of the                NADH/NADPH oxidase p22 phox gene;            -   (4) polymorphism at the base number position −6 of the                angiotensinogen gene:            -   (5) polymorphism at the base number position −219 of the                apolipoprotein E gene;            -   (6) polymorphism at the base number position 994 of the                platelet-activating factor acetylhydrolase gene;            -   (7) polymorphism at the base number position −482 of the                apolipoprotein C-III gene;            -   (8) polymorphism at the base number position 1186 of the                thrombospondin,4 gene;            -   (9) polymorphism at the base number position −819 of the                interleukin-10 gene; and            -   (10) polymorphism at the base number position −592 of                the interleukin-10 gene;        -   (ii) determining, based on the information about            polymorphism which was obtained in the step (i), the            genotype of the nucleic acid sample; and        -   (iii) assessing, based on the genotype determined, a genetic            risk of myocardial infarction.    -   14. A method for diagnosing the risk of myocardial infarction,        comprising the following steps (iv) to (vi):        -   (iv) a step of analyzing the following polymorphisms (11)            to (15) in a nucleic acid sample:            -   (11) polymorphism at the base number position −1171 of                the stromelysin 1 gene;            -   (12) polymorphism at the base number position −668 of                the plasminogen activator inhibitor-1 gene;            -   (13) polymorphism at the base number position 1018 of                the glycoprotein Ibα gene;            -   (14) polymorphism at the base number position 584 of the                paraoxonase gene: and            -   (15) polymorphism at the base number position 4070 of                the apolipoprotein E gene.        -   (v) determining, based on the information about polymorphism            which was obtained in the step (iv), the genotype of the            nucleic acid sample; and        -   (vi) assessing, based on the genotype determined, a genetic            risk of myocardial infarction.

INDUSTRIAL APPLICABILITY

According to the present invention, gene polymorphisms associated withmyocardial infarction are analyzed and the genotypes of nucleic acidsample are detected. By using the information about polymorphismsobtained by the detection of the genotypes, diagnosis of the risk ofmyocardial infarction with high accuracy and high predictability can becarried out. Therefore, the present invention is an effective means forunderstanding the risk of development of myocardial infarction inadvance. Furthermore, according to the present invention, auxiliaryinformation useful for diagnosing the disease is obtained, thus enablingmore appropriate treatment and improvement of prognosis. In addition,the present invention provides information useful in clarifying thedevelopment mechanism of myocardial infarction and is expected tocontribute to prevention and treatment of myocardial infarction.

1. A method for diagnosing the risk of myocardial infarction, comprisingthe following steps (i) to (iii): (i) analyzing two polymorphisms (1)and (2) in a nucleic acid sample: (1) polymorphism at the base numberposition 1019 of the connexin 37 gene; and (2) polymorphism at the basenumber position 242 of the NADH/NADPH oxidase p22 phox gene; (ii)determining, based on the information about said polymorphism which wasobtained in the step (i), the genotype of the nucleic acid sample; and(iii) assessing, based on the genotype determined, a genetic risk ofmyocardial infarction.